PA-001
Scalable biosynthesis of quantum dots: evolution of size selectivity, solubility and
extracellular production
Bryan Berger1, Zhou Yang2, Leah Spangler1, Victoria Berard1, Qian He2, Li Lu2, Robert
Dunleavy1, Christopher Kiely2, Steven McIntosh1
1Department of Chemical and Biomolecular Engineering, Lehigh University, 2Department
of Materials Science and Engineering, Lehigh University
Biological systems have evolved several unique mechanisms to produce inorganic nanomaterials
of commercial interest. Furthermore, bio-based methods for nanomaterial synthesis
are inherently “green”, enabling low-cost and scalable production of nanomaterials
under benign conditions in aqueous solutions. However, achieving regulated control
of the biological processes necessary for reproducible, scalable biosynthesis of nanomaterials
remains a central challenge. This is especially true of quantum dots (QDs), which
are nanocrystals made from seminconducting metals whose diameter is smaller than the
size of its exciton Bohr radius, leading to size-dependent changes in their optical
properties. Several studies have described production of QDs from biological systems,
but without control over particle size or composition. In this work, we describe the
isolation, selection and characterization of a bacterial system capable of regulated,
extracellular biosynthesis of metal sulfide QDs with extrinsic control over nanocrystal
size. Using directed evolution, we isolated and engineered a bacterial strain (SMCD1)
to (1) exhibit enhanced tolerance against aqueous cadmium acetate (2) produce soluble,
extracellular nanocrystals and (3) regulate nanocrystal size by varying growth conditions.
We estimate yields on the order of grams per liter from batch cultures under optimized
conditions, and are able to reproduce the entire size range of CdS QDs described in
literature. Furthermore, we are able to generalize this approach to not only cadmium,
but PbS QDs as well. Investigation of purified QDs using ESI-MS reveals several putative
proteins that may be involved in biosynthesis, and current work is aimed at improving
photoluminescent properties as well as long-term aqueous stability. Nonetheless, our
approach clearly demonstrates the ability of biological systems to produce advanced,
functional nanomaterials, and provides a template for engineering biological systems
to high-value materials such as QDs at cost and scale.
This work was supported by the National Science Foundation (EFRI-1332349).
PA-002
Protein and Cellular Engineering Platform for Selective and Inducible Apoptotic Proteolysis
Charlie Morgan1,2,3, Juan Diaz3, Jim Wells3
1Chemistry and Chemical Biology Graduate Program, UCSF, 2Pharmaceutical Chemistry
Department, UCSF, 3Molecular and Cellular Pharmacology, UCSF
Proteolysis is a fundamental process in biology; it plays a crucial role across development
of multicellular organisms, aids in maintaining tissue homeostasis, and is integral
in cell signaling. Intracellular proteolysis frequently focuses on proteasome mediated
protein degradation, however the tightly regulated and selective proteolysis mediated
by the cysteine-aspartyl specific proteases, caspases, leave their substrates intact.
The growing list of caspase substrates now tops 1500 proteins; a key unmet question
is to differentiate how individual substrate cleavages directly lead to the profound
morphological transformations that are the hallmark of apoptotic cells. We employ
an optimized site-specific and inducible split-protein protease to examine the role
of a classic apoptotic node, the Caspase Activated DNase (CAD). We describe our engineering
platform of post-transcriptional gene replacement (PTGR), where-by endogenous bi-allelic
ICAD is knocked down and simultaneously replaced with an engineered allele that is
susceptible to cleavage by our engineered TEV protease. Remarkably, selective activation
of CAD alone does not induce cell death, although hallmarks of DNA damage are detected
in human cancer cell lines. Additionally, we show the utility of our technology in
deciphering synthetic lethality resulting from coordinated proteolysis of caspase
substrates that control the apoptotic hallmark of chromatin fragmentation.
PA-003
Improving microbial medium-chain fatty acid production using GPCR-based chemical sensors
Stephen Sarria1, Souryadeep Bhattacharyya2, Pamela Peralta-Yahya1
1 School of Chemistry and Biochemistry, Georgia Institute of Technology, 2School of
Chemical and Biomolecular Engineering, Georgia Institute of Technology
Increasing energy needs have accelerated the demand for renewable alternatives to
petroleum-based fuels; engineered microbes for the production of biofuels have the
potential to fulfill these energy needs. Fatty acids are the immediate precursors
to the advanced biofuels fatty acid methyl esters (FAMEs), which can serve as a “drop
in” replacement for D2 diesel. FAMEs derived from medium-chain fatty acids (C8-C12)
have been shown to have better cold properties than traditional FAMEs (C16-C22). Here,
we engineer a yeast strain for the production of medium chain fatty acids by screening
different thioesterases. Our next goal is to couple a medium-chain fatty acid producing
yeast strain to our previously developed medium-chain fatty acid GPCR-based sensor,
in order to engineer a yeast strain with improved medium-chain fatty acid production
via directed evolution.
PB-001
Applications of 19F-NMR to study protein-ligand interactions and protein conformational
changes in solution
Martine I. Abboud1, Jurgen Brem1, Rasheduzzaman Chowdhury1, Ivanhoe K. H. Leung2,
Timothy D. W. Claridge1, Christopher J. Schofield1
1University of Oxford, Department of Chemistry, 2University of Auckland, School of
Chemical Sciences
Nuclear magnetic resonance (NMR) is a powerful biophysical method for studying protein-ligand
interactions in solution and elucidating the mechanism of action of potential inhibitors.
However, protein NMR can be complicated by the overlap of 1H and other resonances,
hence the resolution needed to assign spectra precisely can be hard to achieve [1].
19F-NMR is increasingly being used to study conformational changes and protein-ligand
interactions in solution because 19F is (i) a spin ½ nucleus, (ii) 100% naturally
abundant, (iii) 83% as sensitive to NMR detection as 1H, (iv) not present in most
biological systems, and (v) its chemical shift is particularly sensitive to changes
in local environment [2]. Recent advancements in NMR instrument and probe design have
made 19F-NMR more sensitive and more widely available; consequently, 19F-NMR is finding
growing application in research. Here, we report the use of 19F-NMR to study two biomedically
important protein systems. Proteins can be fluorine-labelled either by biological
incorporation of fluorinated amino acids or by site-specific chemical ligation [3].
3-bromo-1,1,1-trifluoroacetone (BTFA) has been developed as a useful reagent for importing
fluorine into proteins via nucleophilic substitution such as with a cysteinyl-thiol
[4]. The São Paulo metallo-β-lactamase-1 (SPM-1), a B1 sub-family metallo-β-lactamase,
containing only one cysteine (Cys221) coordinating the second Zn(II) cation in its
active site, was 19F-labelled using BTFA. The interactions of SPM-1 with various potential
inhibitors were reported by 19F-NMR, which enabled monitoring SPM-1 conformational
changes on ligand binding and informed on binding strength by enabling KD measurements.
In a second study, prolyl hydroxylase 2 (PHD2), an enzyme involved in human oxygen
sensing, was 19F-labelled and its interactions with its co-substrate, 2-oxoglutarate
(2OG), and peptide substrate (CODD) were monitored by 19F-NMR. Conformational changes
of PHD2 on 2OG and CODD binding were consistent with crystallographic analyses. Finally,
19F-NMR was used to study solvent exposure of the complex and its dynamics in solution
through relaxation dispersion of the 19F-nucleus at different temperatures. Overall,
the results illustrate the power of 19F-NMR for monitoring ligand binding and conformational
changes.
[1] Wemmer, B. D. E., & Williams, P. G. (1994). Use of nuclear magnetic resonance
in probing ligand-macromolecule. Methods in Enzymology.
[2] Marsh, E. N. G., & Suzuki, Y. (2014). Using 19F NMR to Probe Biological Interactions
of Proteins and Peptides. ACS Chemical Biology.
[3] Chen, H., Viel, S., Ziarelli, F., & Peng, L. (2013). 19F NMR: a valuable tool
for studying biological events. Chemical Society Reviews.
[4] Rydzik, A. M., Brem, J., van Berkel, S. S., Pfeffer, I., Makena, A., Claridge,
T. D. W., & Schofield, C. J. (2014). Monitoring conformational changes in the NDM-1
metallo-β-lactamase by 19F-NMR spectroscopy. Angewandte Chemie.
PB-002
NMR solution structure of lacticin Q, a broad spectrum leaderless antimicrobial protein
from Lactococcus lactis QU 5
Jeella Acedo1, Marco van Belkum1, John Vederas1
1Department of Chemistry, University of Alberta
Bacteriocins are ribosomally synthesized antimicrobial peptides and proteins that
can be potentially used as food preservatives and are promising alternatives to traditional
antibiotics. Lacticin Q from Lactococcus lactis QU 5 belongs to an unusual class of
bacteriocins classified by the absence of an N-terminal leader sequence. It is composed
of 53 amino acids and has previously been reported to be active against a broad spectrum
of Gram-positive bacteria in the nanomolar range. In this study, lacticin Q was expressed
as a recombinant protein fused to SUMO (small ubiquitin-related modifier) protein.
After cleavage of the SUMO-tag and subsequent purification, mass spectrometry was
used to confirm the identity of the recombinant lacticin Q. Its three-dimensional
nuclear magnetic resonance (NMR) solution structure was then elucidated. Both circular
dichroism and NMR spectroscopy results reveal that lacticin Q is highly α-helical.
It has a compact, globular overall fold and has a cationic surface and a hydrophobic
core. These structural data support previously established mechanism of action, whereby
antimicrobial activity is attributed to the binding of lacticin Q to anionic bacterial
cell membrane and subsequent formation of pores that result in the leakage of cell
contents. The elucidated structure of lacticin Q resembles the two-component leaderless
bacteriocins enterocins 7A and 7B, which are 9- and 10- amino acids shorter than lacticin
Q. This suggests that the observed overall fold might be conserved among this class
of bacteriocins.
PB-003
Sizing and interactions of proteins under native conditions from microfluidic diffusion
measurements: application to molecular chaperones and single-step immunoassay
Paolo Arosio1, Thomas Müller1, Luke Rajah1, Francesco Aprile1, Tom Scheidt1, Jackie
Carrozza1, Maya Wright1, Michele Vendruscolo1, Christopher Dobson1, Tuomas Knowles1
1Department of Chemistry, University of Cambridge
Characterizing the sizes and shapes of proteins and their interactions is of fundamental
importance for understanding the behavior of a large variety of systems in the biological
and biotechnological sciences. Defining these properties under native conditions,
directly in solution and on a second timescale, remains, however, challenging. To
address this problem, we have developed a method based on monitoring micron-scale
diffusion in both space and time by acquiring, in a microfluidic format, diffusion
profiles at different diffusion times under steady-state flow conditions. We show
that the global analysis of this combined space-time acquisition enables the average
sizes of the components of monodisperse and polydisperse solutions, as well as the
sizes of individual species within binary mixtures, to be determined directly. We
show that the ability to perform rapid and non-invasive sizing enables this technique
to be used to quantify the thermodynamics and the kinetics of specific interactions
between molecular chaperones and protein aggregates in complex polydisperse solutions,
as well as to identify the oligomerization state of dynamic protein systems. We demonstrate
further a quantitative immunoassay that enables specific interactions between biomolecules
as well as the conformations of target protein species to be determined directly in
solution even in heterogeneous mixtures.
PB-004
Using α-chymotrypsin and elastase enzymatic degradation to control peptide self-assembly
Valeria Castelletto1, Ian Hamley1
1School of Chemistry, University of Reading
A micellar nanocontainer delivery and release system is designed on the basis of a
peptide-polymer conjugate. 1 The hybrid molecules self-assemble into micelles comprising
a modified amyloid peptide core surrounded by a PEG corona. The modified amyloid peptide
previously studied in our group forms helical ribbons based on a β-sheet motif and
contains β-amino acids that are excluded from the β-sheet structure, thus being potentially
useful as fibrillization inhibitors. In the model peptide-PEG hybrid system studied,
enzymatic degradation using R-chymotrypsin leads to selective cleavage close to the
PEG-peptide linkage, break up of the micelles, and release of peptides in unassociated
form. The release of monomeric peptide is useful because aggregation of the released
peptide into β-sheet amyloid fibrils is not observed. This concept has considerable
potential in the targeted delivery of peptides for therapeutic applications. In a
separate work, the self-assembly of the alanine-rich amphiphilic peptides Lys(Ala)6Lys
(KA6K) and Lys(Ala)6Glu (KA6E) with homotelechelic or heterotelechelic charged termini
respectively has been investigated in aqueous solution. 2 These peptides contain hexa-alanine
sequences designed to serve as substrates for the enzyme elastase. Electrostatic repulsion
of the lysine termini in KA6K prevents self-assembly, whereas in contrast KA6E is
observed, through electron microscopy, to form tape-like fibrils, which based on X-ray
scattering contain layers of thickness equal to the molecular length. The alanine
residues enable efficient packing of the side-chains in a β-sheet structure, as revealed
by circular dichroism, FTIR and X-ray diffraction experiments. In buffer, KA6E is
able to form hydrogels at sufficiently high concentration. These were used as substrates
for elastase, and enzyme-induced de-gelation was observed due to the disruption of
the β-sheet fibrillar network. We propose that hydrogels of the simple designed amphiphilic
peptide KA6E may serve as model substrates for elastase and this could ultimately
lead to applications in biomedicine and regenerative medicine.
[1] V. Castelletto; J. E. McKendrick; I. W. Hamley; U. Olsson; C. Cenker, Langmuir,
2010, 26, 11624.
[2] V. Castelletto; R. J. Gouveia; C. J. Connon; I. W. Hamley; J. Seitsonen; J. Ruokolainen;
E. Longo; G. Siligardi, Biomaterials Science, 2014, 2, 867.
PB-005
Fluorescence-based techniques for the investigation of localization and functions
of proteins
Yuen-Yan Chang1, Yau-Tsz Lai1, Ligang Hu1, Ya Yang1, Ailun Chao1, Hongzhe Sun1
1Department of Chemistry, The University of Hong Kong
Two fluorescence-based techniques are applied to label intracellular tagged proteins
and monitor the interaction between a metalloprotein and Bi-based metallodrugs. First,
we have synthesized, to our knowledge, the first membrane permeable fluorescent probe
Ni-NTA-AC that enters cells and monitors intracellular His-tagged proteins in minutes,
without functionally perturbing the target proteins (Figure 1) [1]. Arylazide photoactivation
to covalently linked the His-tagged proteins by Ni-NTA-AC resulted in significant
fluorescence enhancement (∼13-fold). Ni-NTA-AC successfully traced the subcellular
localization of His-tagged proteins with negligible toxicity in different biological
systems, including bacterial and mammalian cells and even the plant tissues. We are
currently developing fluorescent probes with different fluorophores using similar
strategy to enable the simultaneous examination of tagged target proteins in cells.
Besides, we have constructed two fluorescent sensors CYHpnl and CYHpnl_1-48 (with
C-terminus glutamine-rich sequence deleted) to elucidate the role of metalloprotein
Hpn-like by Fluorescence Resonance Energy Transfer (FRET) (Figure 2) [2]. We found
the selective coordination of Ni(II) and Zn(II) to the purified sensors and in E.
coli cells. Surprisingly, specific interaction between the FRET sensors and Bi(III)
was observed. Our FRET analysis confirmed the role of Hpnl for Ni(II) storage and
revealed the potential association of Hpnl with Bi-based antiulcer drugs in cells.
Figure 1
Figure 2
This work was supported by the Research Grants Council of Hong Kong (704909 and N_HKU75209,
704612P, 703913P), Livzon Pharmaceutical Group and the University of Hong Kong (for
the emerging Strategic Research Theme – Integrative Biology).
[1] Y.T. Lai, Y. Y. Chang, L. Hu, Y. Yang, A. Chao, Z. Y. Du, J. A. Tanner, M. L.
Chye, C. Qian, K. M. Ng, H. Li, H. Sun, Proc. Natl. Acad. Sci. USA, 2015, 112, 2948–2953
[2] Y. Y. Chang, Y. T. Lai, T. Cheng, H. Wang, Y. Yang, H. Sun, J. Inorg. Biochem.,
2015, 142, 8–14
PB-006
RNA Fate is Controlled by Highly-Regulated RNA Binding Proteins
Irene Díaz-Moreno1, Isabel Cruz-Gallardo1, Sofía M. García-Mauriño1, Rebecca Del Conte2,
B. Göran Karlsson3, Andres Ramos4, María L. Martínez-Chantar5, Francisco J. Blanco5,
Myriam Gorospe6, Jacqueline A. Wilce7
1IBVF - cicCartuja, University of Seville - CSIC, 2CERM, Department of Chemistry,
University of Florence, 3Swedish NMR Centre, University of Gothenburg, 4Molecular
Structure Division, MRC National Institute for Medical Research, 5CIC bioGUNE, 6Laboratory
of Genetics, National Institute on Aging-Intramural Research Program, 7Department
of Biochemistry and Molecular Biology, Monash University
RNA biology is tightly orchestrated by the interplay of RNAs with RNA-Binding Proteins
(RBPs), which can be regulated by post-translational modifications, pH-dependence
and oligomerization states. Phosphorylation of the RBP K-Homology Splicing Regulatory
Protein (KSRP) induces protein unfolding and impairs the ability of KSRP to promote
the degradation of its RNA targets [1,2]. This finding reveals the molecular mechanism
that links the mRNA-degradation pathway with extracellular signaling networks through
the reversible unfolding of a RNA binding domain (RBD). RNA binding is also controlled
by pH conditions. This finding becomes relevant for RBPs such as T-cell Intracellular
Antigen 1 (TIA-1), which shuttles between two cellular compartments (nucleus and cytoplasm)
with slightly different pH values. In fact, RNA binding by TIA-1 is modulated by slight
environmental pH changes due to the protonation/deprotonation of TIA-1 histidine residues
[3,4]. The pH dependence of the TIA-1/RNA interaction provides a new insight into
the function of TIA-1 in recognizing new RNA targets [5], like the 5’ Terminal Oligopyrimidine
Tracts (5´TOPs) of translationally-repressed mRNAs. Along with TIA-1, the RBP Hu antigen
R (HuR) is involved in the assembly/disassembly of cytoplasmic Stress Granules (SG),
which arise as a protective mechanism by preventing mRNA decay under stress situations.
Despite wide acceptance that RBPs harboring aggregation-promoting Prion Related Domains
(PRDs), such as TIA-1, stimulate rapid self-association and formation of SGs, we propose
that scaffolding SGs may be driven by RBDs, since PRD-lacking RBPs, like HuR, often
form oligomers [6,7,8] and are included in SGs. Under continuous stress, the transition
from the physiological to pathological aggregation of RBPs in SGs may depend on post-translational
modifications of RBDs. RNA-binding proteinopathies, characterized by the nucleation
of irreversible SGs, are often found in neurodegenerative diseases. Altogether, resulting
insights into RNA biology suggest that highly-regulated RBPs determine mRNA fate from
synthesis to decay.
[1] Díaz-Moreno et al. (2009) Nat. Struct. Mol. Biol. 16: 238-246
[2] Díaz-Moreno et al. (2010) Nucleic Acids Res. 38: 5193-5205
[3] Cruz-Gallardo et al. (2013) J. Biol. Chem. 288: 25986-25994
[4] Cruz-Gallardo et al. (2015) Eur. Chem. J. in press
[5] Cruz-Gallardo et al. (2014) RNA Biol. 11: 766-776
[6] Scheiba et al. (2012) Eur. Biophys. J. 41: 597-605
[7] Scheiba et al. (2014) RNA Biol. 11: 1250-1261
[8] Díaz-Quintana et al. (2015) FEBS Lett. in press
PB-007
Understanding promiscuous and selective ligand binding by liver FABP
Mariapina D’Onofrio1, Filippo Favretto1, Serena Zanzoni1, Silvia Perez Santero1, Michael
Assfalg1, Henriette Molinari2, Carlo Santambrogio2, Rita Grandor2
1Department of Biotechnology, University of Verona, Strada Le Grazie 15, 2Laboratorio
NMR, ISMAC-CNR
Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules,
and play a role in global metabolism regulation. Liver FABP (L-FABP) is characterized
by high versatility in terms of ligand binding capabilities.[1] Indeed, both long
chain fatty acids as well as bulkier ligands can be accommodated into the protein’s
large internal cavity. The involvement of L-FABP in the transport of bile salts has
been postulated but scarcely investigated.[2] This hypothesis is further supported
by the realization that L-BABP (a type 2 intracellular lipid binding protein bile-salt
carrier) is absent in mammals. We have used a variety of NMR experiments, as well
as steady-state fluorescence spectroscopy, and mass spectrometry to gain insight,
at molecular and atomic level, into the interactions established by human L-FABP with
a pool of bile acids [3, 4], contributing to improve our understanding of the binding
specificity for this important class of cholesterol-derived metabolites. An extensive
comparison among L-FABP alone, in complex with bile acids, and in complex with oleate,
has been performed in order to investigate the distinctive features of L-FABP binding
promiscuity.[3] NMR relaxation experiments on different timescales suggest that human
L-FABP is poorly selective in terms of ligand binding and a functional role is played
by its internal dynamics. Taken together our findings expand the current knowledge
about ligand recognition by L-FABP with implications in the intracellular transport
of bile acids in physiological and pathological states.
[1] Zimmerman AW, Veerkamp JH (2002) Cell Mol Life Sci. 59, 1096–1116.
[2] Guariento M, Raimondo D, Assfalg M, Zanzoni S, Pesente P, Ragona L, Tramontano
A & Molinari H (2008) Proteins 70, 462–472.
[3] Favretto F, Assfalg M, Gallo M, Cicero DO, D’Onofrio M & Molinari H (2013) ChemBioChem
14, 1807–1819.
[4] Favretto F, Santambrogio C, D’Onofrio M, Molinari H, Grandori R & Assfalg M (2015)
FEBS J. 282,1271–88.
PB-008
Antimalarial Agents With a Novel Mode of Action: Dual Inhibition of P. falciparum
M1 and M17 Metalloaminopeptidases
Nyssa Drinkwater1, Shailesh Mistry2, Komagal Kannan Sivaraman1, Alessandro Paiardini3,
Vicky Avery4, Peter Scammells2, Sheena McGowan1
1Department of Biochemistry & Molecular Biology, Monash University, 2Monash Institute
of Pharmaceutical Sciences, Monash University, 3Dipartmento di Scienze Biochimiche,
Sapienza Universita di Roma, 4Eskitis Institute for Drug Discovery, Griffith University
Malaria is caused by parasites of the genus Plasmodium, with P. falciparum (Pf) causing
the most fatalities. The prevention and treatment of Pf malaria is becoming increasingly
difficult due to the spread of drug resistant parasites. New therapeutics with a novel
mode of action are desperately required. Two P. falciparum aminopeptidases, PfA-M1
and PfA-M17, play crucial roles in the erythrocytic stage of infection, and have been
validated as potential antimalarial targets. Using compound-bound crystal structures
of both enzymes, we were able to identify key similarities and differences in the
mechanism of inhibitor binding by PfA-M1 versus PfA-M17, which we exploited to design
inhibitors capable of potently inhibiting both enzymes. The resultant hydroxamic acid-based
inhibitors represent the first compounds capable of potent dual inhibition of both
PfA-M1 and PfA-M17. The compounds additionally possess nanomolar activity against
3D7 malaria parasites and no observable cytotoxicity, and are therefore extremely
attractive lead molecules for further development into antimalarial therapeutics with
a novel mode of action.
PB-009
Different classes of inhibitors for human 15-lipoxygenase-1
Nikolaos Eleftheriadis1, Stephanie Thee1, Johan te Biesebeek1, Petra van der Wouden1,
Bert-Jan Baas1, Frank J. Dekker1
1Groningen Research Institute of Pharmacy
Lipoxygenases metabolize polyunsaturated fatty acids into signalling molecules such
as leukotrienes and lipoxins. 15-lipoxygenase (15-LOX) is an important mammalian lipoxygenase
and plays a crucial regulatory role in several respiratory diseases such as asthma,
COPD and chronic bronchitis. Novel potent and selective inhibitors of 15-LOX-1 are
required to explore the role of this enzyme in drug discovery. In this study we present
different classes of inhibitors for human 15-LOX-1. Kinetic analysis suggests competitive
inhibition and the binding model of these compounds can be rationalized using molecular
modelling studies. The new inhibitors show Ki values from 0.040 µM to 1.7 µM. These
structure-activity relationships provide a basis to design improved inhibitors and
explore 15-LOX-1 as a drug target.
PB-010
Identification of Novel Inhibitors of 6-Phosphogluconate Dehydrogenase (6PGDH) in
Trypanosoma brucei Through Virtual Drug Screening
Victoria Gomez1, Kavya Kolavasi1, Josh Beckham1, Jon Robertus1
1The University of Texas at Austin College of Natural Science
A threat to 70 million people in underdeveloped nations around the world, African
trypanosomiasis (sleeping sickness) is a neglected tropical disease (NTD) caused by
the protozoan parasite Trypanosoma brucei (T. brucei). T. brucei is transmitted to
humans via the tsetse fly, and replicates in the blood before crossing into the brain,
causing death for the infected individual. Current treatments that are available for
African sleeping sickness are highly toxic and usually difficult to administer past
the blood-brain barrier. It is our belief that coupling less toxic compounds with
efficient drug delivery systems will contribute to the development of the most effective
drug against African sleeping sickness. Our goal was to determine a novel and effective
chemical inhibitor with the potential to prevent the replication of T. brucei in the
human body. The enzyme target for inhibition studied in this research was 6-phosphogluconate
dehydrogenase (6PGDH), a cytosolic enzyme in the pentose phosphate pathway (PPP) of
T. brucei. 6PGDH is essential in the PPP due to its ability to oxidize 6-phosphogluconate
into ribulose-5-phosphate, which is essential for the formation of nucleotides. Primer
overlap extension Polymerase Chain Reaction (PCR) was used to synthesize the coding
DNA sequence of the 6PGDH gene, which was then cloned into a pNIC-Bsa4 inducible expression
plasmid with an N-terminal 6 Histidine tag, by way of ligation independent cloning.
The protein was then expressed in BL21 (DE3) Escherichia coli (E. coli) cells and
purified via nickel column affinity and size exclusion fast protein liquid chromatography
(FPLC) to perform inhibition assays. Through virtual screening, various ligands obtained
from the Chembridge Library and NIH Clinical Collection) were docked into the active
site of the crystal structure of Tb6pgdh (Pubchem identification 1PGJ) using GOLD
molecular docking software. The top scoring compounds were selected by utilizing parameters
such as hydrophobic interactions, hydrogen bonds, and Van der Waals forces. The compounds
with the best scores that also satisfied Lipinski’s Rule of 5 criteria for druggability
were then tested in spectrophotometric enzyme inhibition assays monitoring the absorbance
of NADPH at 340 nm. Compounds that show inhibitory activity in the assays will be
taken to higher levels of testing to determine their effect on T. brucei in other
organisms.
PB-011
NMR studies of the structural influence of phosphopantetheinylation in nonribosomal
peptide synthetase carrier proteins and impact on binding affinities
Andrew Goodrich1, Dominique Frueh1
1Johns Hopkins University School of Medicine
Nonribosomal peptide synthetases (NRPSs) are modular enzymatic systems responsible
for the production of complex secondary metabolites in bacteria and fungi. Each module
is comprised of (at least) three core domains whose combined action leads to the selection,
activation, and incorporation of a single small molecule into a growing peptide. Central
to each module is the carrier protein (CP), which is first primed via attachment of
a 4’-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active
holo form. An adenylation (A) domain then covalently attaches an amino or aryl acid
onto the ppant arm via formation of a thioester. The CP then shuttles activated monomers
and growing peptides between the active sites of catalytic domains in both the same
and adjacent modules. During CP priming and peptide elongation, a CP thus exists in
multiple different post-translational states and interacts with numerous catalytic
domains. Understanding how NRPSs are able to efficiently orchestrate this series of
sequential protein-protein interactions between a CP and its partner catalytic domains
is key to unraveling the molecular mechanism of NRP synthesis. Using a combination
of isothermal titration calorimetry and nuclear magnetic resonance (NMR) titrations,
we found that converting a CP from the apo to holo form alters its affinity for its
partner A domain. This change in binding suggests a means by which directionality
in protein-protein interactions is achieved in NRPSs. However, we also found that
A domain binding affects the same subset of residues in both the apo and holo forms.
In order to identify the molecular features underpinning this difference in affinity,
we solved the NMR solution structures of the apo and holo forms of the CP. Here, we
present the solution structures of an apo and holo CP and discuss them in light of
their differential binding to an A domain.
PB-012
Functional analysis of of conditional analog-sensitive alleles of essential protein
kinases in the fission yeast Schizosaccharomyces pombe.
Juraj Gregan1, 2
1Mfpl/imp, 2Comenius University
The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases
that are essential for viability. Studies of the essential kinases often require the
use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally,
we applied a recently developed chemical genetic strategy. The mutation of a single
residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors,
allowing for specific inactivation of the modified kinase. Using this approach, we
constructed conditional analog-sensitive alleles of 13 essential protein kinases in
the fission yeast S. pombe. I will present the functional analysis of these mutants
during meiosis.
PB-013
Peptide conjugates: From self-assembly towards applications in biomedicine
Ian Hamley1
1University Of Reading, Dept of Chemistry
Self-assembling peptides and their conjugates offer exceptional potential in nanomedicine.
I will present some of our recent work on nanoscale assembled peptides and their conjugates,
focussing on lipopeptides [1, 2] and PEG-peptide conjugates [3]. PEGylation is an
important technique in the development of conjugates for applications in therapeutics.
It is found to greatly influence self-assembly of peptides and proteins - one example
from our own work is a peptide which itself forms twisted fibrils but when PEG is
attached, self-assembly of the conjugate leads to spherical micelles[4]. The conjugate
can be enzymatically degraded using alpha-chymotrypsin, releasing the peptide. This
nanocontainer delivery and release system could be useful in therapeutic applications.
Thermoresponsive telechelic PEG/peptides with hydrophobic dipeptide end groups (di-tyrosine
or di-phenylalanine) were developed, one of which shows a de-gelation transition near
body temperature and which may be useful in bioresponsive delivery systems [5]. Examples
from our recent work on self-assembling lipopeptides will also be outlined. Our focus
is to investigate potential relationships between self-assembly and bioactivity, in
particular in the fields of regenerative medicine [6-10], antimicrobial systems [11,
12] and immune therapies [13].
[1] Dehsorkhi A, Castelletto V, Hamley IW. Self-Assembling Amphiphilic Peptides. J.
Pept. Sci. 2014;20:453-67.
[2] Hamley IW. Self-Assembly of Amphiphilic Peptides. Soft Matter 2011;7:4122-38.
[3] Hamley IW. PEG-Peptide Conjugates. Biomacromolecules 2014;15:1543-59.
[4] Castelletto V, McKendrick JME, Hamley IW, Cenker C, Olsson U. PEGylated Amyloid
Peptide Nanocontainer Delivery and Release System. Langmuir 2010;26:11624-27.
[5] Hamley IW, Cheng G, Castelletto V. Self-Assembly of Telechelic PEG End-capped
with Hydrophobic Dipeptides. Macromol. Biosci. 2011;11:1068-78.
[6] Jones RR, Castelletto V, Connon CJ, Hamley IW. Collagen Stimulating Effect of
Peptide Amphiphile C16-KTTKS on Human Fibroblasts. Mol. Pharm. 2013;10:1063-69.
[7] Castelletto V, Hamley IW, Whitehouse C, Matts P, Osborne R, Baker ES. Self-Assembly
of Palmitoyl Lipopeptides Used in Skin Care Products. Langmuir 2013;29:9149-55.
[8] Gouveia RJ, Castelletto V, Alcock SG, Hamley IW, Connon CJ. Bioactive films produced
from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion
and tissue architecture in serum-free conditions. Journal of Materials Chemistry B
2013;1:6157-69.
[9] Castelletto V, Gouveia RJ, Connon CJ, Hamley IW, Seitsonen J, Ruokolainen J, Longo
E, Siligardi G. Influence of elastase on alanine-rich peptide hydrogels. Biomaterials
Science 2014;2:867-74.
[10] Gouveia RJ, Castelletto V, Connon CJ, Hamley IW. Submitted 2014.
[11] Dehsorkhi A, Castelletto V, Hamley IW, Seitsonen J, Ruokolainen J. Interaction
Between a Cationic Surfactant-Like Peptide and Lipid Vesicles and Its Relationship
to Antimicrobial Activity. Langmuir 2013;29:14246-53.
[12] Hamley IW, Dehsorkhi A, Castelletto V. Self-Assembled Arginine-Coated Peptide
Nanosheets in Water. Chem. Comm. 2013;49:1850-52.
[13] Hamley IW, Kirkham S, Dehsorkhi A, Castelletto V, Reza M, Ruokolainen J. Toll-like
Receptor Agonist Lipopeptides Self-Assemble into Distinct Nanostructures. Chem. Comm.
2014; 50: 15948-51.
PB-014
Approved Drugs containing Thiols as Inhibitors of Metallo-ß-Lactamases: A Strategy
to Combat Multidrug-Resistant Bacteria
Franca-M. Klingler1, Ewgenij Proschak1
1Goethe University, Institute of Pharmaceutical Chemistry
Antibiotic resistance in bacterial pathogens is one of the major threats regarding
human health. An alarming trend is the spread of metallo-β-lactamases (MBLs) among
gram-negative pathogens that transfer resistance against almost all β-lactams including
carbapenems. [1] The development of new anti-infective agents remains one of the most
significant demands in modern medicine. [2] The aim of this work is to find an already
approved drug which restores the activity of β-lactam antibiotic by protecting it
from hydrolysis through the MBL. Thiol groups are known zinc chelators and therefore
inhibit MBLs.[3] We established a novel sensitive fluorescence-based assay platform
for studying inhibition of β-lactamases using the commercially available substrate
Fluorocillin.[4] The reliability of the system was evaluated on three different class
B MBLs: New-Delhi-Metallo- β-Lactamase-1 (NDM-1), Verona-Integron-Encoded-Metallo-β-Lactamase
1 (VIM-1) and Impenemase-7 (IMP-7). Remarkably, not all compounds inhibited MBLs,
although every compound carried a thiol group. In order to discriminate between zinc-withdrawing
and direct binding to the enzyme, thermal shift assay was conducted. This assay combination
provides a method to find inhibitors which inhibit MBLs via direct binding to the
active site.[5] Most promising compounds were passed to antimicrobial susceptibility
testing using laboratory strains and patient isolates. Results showed that some of
our compounds partially restored the efficacy of Imipenem against pathogenic bacteria.
Overall, we found four approved drugs, which inhibit three clinically important MBLs,
namely Captopril, Thiorphan, Dimercaprol and Tiopronin. This result yields a good
starting point for the development of potent MBL inhibitors, with the primary optimization
goal being the uptake and activity in pathogens.
[1] M. a Fischbach, C. T. Walsh, Science 2009, 325, 1089–1093.
[2] H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Edwards, D. Gilbert, L. B. Rice,
M. Scheld, B. Spellberg, J. Bartlett, Clin. Infect. Dis. 2009, 48, 1–12.
[3] C. Bebrone, Biochem. Pharmacol. 2007, 74, 1686–1701.
[4] A. Rukavishnikov, K. R. Gee, I. Johnson, S. Corry, Anal. Biochem. 2011, 419, 9–16.
[5] F. H. Niesen, H. Berglund, M. Vedadi, Nat. Protoc. 2007, 2, 2212–21.
PB-015
Protein Carbamylation at the Chemistry-Biology interface
Victoria Linthwaite1, Joana Janus1, David R.W. Hodgson2, Martin J. Cann1
1School of Biological and Biomedical Sciences, Durham University, 2Department of Chemistry,
Durham University
Carbon dioxide (CO2) is a crucial regulator for all three domains of life (1), known
for its role during respiration and photosynthesis. Despite this there is very little
known about its molecular interactions with cellular components. CO2 combines rapidly
but reversibly with amines at physiological temperatures and pressures to form carbamates
(2). This modification is present in key proteins, such as RuBisCO and haemoglobin
but remains unexplored in other systems. Carbamylation is caused by the nucleophilic
attack of an uncharged amine (for example on an arginine or lysine side chain or an
N-terminal group) on CO2 (2). This research aims to investigate this understudied
modification.
We are resolving these limitations to investigate carbamate formation in cellular
systems by trapping carbamates chemically thereby removing their labile nature3. We
are developing chemical and analytical tools to meet this challenge. Main results:
We have successfully proven the ability to trap carbamates on acetyl-lysine, Lys-Gly
and Phe-Gly dipeptides, a tetra-peptide and haemoglobin. These results have been confirmed
using ESI-MS combined with 12C and 13C isotope incorporation. Further work using radioactive
14C has been carried out on whole organism samples.
1. Tang, X. D., et al., (2004) Metabolic regulation of potassium channels. Annu. Rev.
Physiol. 66, 131-159
2. Hampe, E. M., and Rudkevich, D. M. (2003) Exploring reversible reactions between
CO2 and amines. Tetrahedron 59, 9619-9625
3. Terrier, P., and Douglas, D. J. (2010) Carbamino Group Formation with Peptides
and Proteins Studied by Mass Spectrometry. Journal of the American Society for Mass
Spectrometry 21, 1500-1505
PB-016
A β-carboline substituted derivative displays selective anti-cancer activity through
inhibition of translation
Annelise de Carvalho1, Jennifer Chu2, Céline Meinguet3, Robert Kiss1, Guy Vandenbussche4,
Bernard Masereel3, Yohan Wouters3, Jerry Pelletier2, Véronique Mathieu1
1Laboratoire de Cancérologie et Toxicologie Expérimentale, Faculté de Pharmacie, 2Biochemistry
Department, 3Namur Medicine and Drug Innovation Center (NAMEDIC-NARILIS), 4Laboratory
for the Structure and Function of Biological Membranes
Background: Reprogrammed cellular metabolism is one of the ten recognized hallmarks
of cancer cells (Hanahan and Weinberg, 2011). More particularly, increased cell proliferation,
migration, angiogenic induction and modifications to the tumor environment require
elevated protein synthesis and turnover. Surprisingly, the only FDA-approved anticancer
drugs that targets protein metabolism in cancer cells are recently launched proteasomal
inhibitors and omacetaxine mepesuccinate, a translation elongation inhibitor. Regulation
of protein synthesis in tumor cells differs significantly from their non-transformed
counterparts due to alterations in MAPK and Akt/mTOR signaling pathways, two major
signal transduction pathways that control initiation of translation. There is thus
significant interest in the development of novel inhibitors of translation as potential
anti-neoplastic agents. Purpose and results: In a previous study, we reported novel
substituted β-carbolines as protein synthesis inhibitors (Frederick and Bruyere et
al, 2012). CM16, the lead compound optimized in terms of pharmacological properties
(Meinguet et al, 2015), was used to start to decipher their mode of action. CM16 exerts
cytostatic anti-cancer effects in vitro without significant modification of the cell
cycle profile. The National Cancer Institute COMPARE algorithm enabled us to compare
the global growth inhibition profile of CM16 on 60 cancer cell lines (mean GI50 of
0.2 µM) to the >765,000 compounds of their database and revealed good correlation
coefficients with protein synthesis inhibitors. Effects of CM16 on transcription could
appear secondary since we didn’t observed any effects before prolonged compound incubation
on cells - at least 24h exposure to 5 µM CM16. In contrast, translation was quite
sensitive to CM16, as assessed by metabolic labeling assays. Ribosomal subunit assembly
and polysome profiling of cells exposed to CM16 were evaluated by sucrose gradient
analysis. Exposure of cells to CM16 for only 3h was sufficient to disrupt polysomes
and cause an increase in 80S complexes. Ongoing investigations of the expression and
phosphorylation status of different initiation and elongation factors identified eIF2α;
as a potential target of CM16. This effect unlikely results from a direct activity
of CM16 on PERK, one of four eIF2α; kinases. CM16 penetrates into the cancer cells
after few minutes of treatment and its distribution parallels an endoplasmic reticulum
fluorescent probe. Interestingly, non-transformed cells are ∼10 times less sensitive
to CM16. Conclusions and Perspectives: CM16 is a synthetic harmine derivative that
displays anti-neoplastic activity in vitro at concentrations of 0.1 to 0.5 µM. It
appears to inhibit protein synthesis at the level of the initiation phase within in
the first 3 hours following exposure to cells. Differences in the dependencies on
deregulated protein synthesis in normal versus transformed cells appear to be exploitable
for the development of new anti-cancer agents. Additional proteomic studies will be
conducted in the near future to further our understanding of this concept and the
effects of CM16.
PB-017
Semi-chemical synthesis and characterization of a small heat shock protein bearing
a nonenzymatic posttranslational modification found in vivo
Maria Matveenko1, Christian Becker1
1Institute of Biological Chemistry, Department of Chemistry, University of Vienna
Up to 50% of all human proteins are believed to be modified following their biosynthesis
through posttranslational modifications (PTMs) [1]. PTMs can result from enzymatic
and nonenzymatic reactions, and both types of modifications play an important role
in a plethora of physiological and pathological processes [2,3]. Nonenzymatic modifications
(NEMs) are increasingly recognized to affect cellular processes, with an involvement
in age-related, metabolic and neurodegenerative diseases [4,5]. Human heat shock protein
(Hsp27) has been shown to become derivatized with argpyrimidine, a prominent NEM that
occurs on arginine residues [6], in certain human cancer tissues and cell lines [7,8].
This NEM was linked to the elevated antiapoptotic activity of the protein [7,8], whereby
modification of Arg-188 appeared to be of particular significance [7]. In this work,
Hsp27 homogeneously modified with argpyrimidine at position 188 is generated for the
first time. Using expressed protein ligation [9], the first semisynthesis of the unmodified
protein is achieved as well. Our approach, which combines organic chemistry, peptide
synthesis and protein synthesis, enables complete control over protein composition
and thus can provide previously unattainable insight into the properties of this vital
chaperone following nonenzymatic modification. The synthesis of argpyrimidine-modified
Hsp27 and the progress towards structural and functional characterization of the protein
will be presented herein.
[1] Khoury, G.A., Baliban, R.C., Floudas, C.A. Sci Rep 2011, 1(139).
[2] Walsh, C.T., Garneau-Tsodikova, S., Gatto, G.J., Jr. Angew Chem Int Ed 2005, 44(45),
7342.
[3] Vistoli G, De Maddis D, Cipak A, Zarkovic N, Carini M, Aldini G. Free Radic Res
2013, 47(Suppl. 1), 3.
[4] Jaisson, S., Gillery, P. Clin Chem 2010, 56(9), 1401.
[5] Rabbani, N., Thornalley, P.J. Amino Acids 2012, 42(4), 1133.
[6] Shipanova, I.N., Glomb, M.A., Nagaraj, R.H. Arch Biochem Biophys 1997, 344(1),
29.
[7] Sakamoto, H., Mashima, T., Yamamoto, K., Tsuruo, T. J Biol Chem 2002, 277(48),
45770.
[8] Van Heijst, J.W., Niessen, H.W., Musters, R.J., van Hinsbergh, V.W., Hoekman,
K., Schalkwijk, C.G. Cancer Lett 2006, 241(2), 309.
[9] Muir, T.W., Sondhi, D., Cole, P.A. PNAS 1998, 95(12), 6705.
PB-018
A new scaffold for inhibition of cysteine proteases: Structural and functional characterization
of Kunitz inhibitors from potato
Manasi Mishra1, Jiri Brynda1, Michael Mares1
1Institute of Organic Chemistry and Biochemistry, AS CR
Kunitz-type protease inhibitors belong to a widespread protein family present in many
plant species and play an important role in plant defense against insect pests and
pathogens. Members of this family are typically inhibitors of proteases of serine
class. Interestingly, a few members were identified as inhibitors of proteases of
cysteine class, however, they have not been functionally and structurally characterized.
Our study is focused on Kunitz-type inhibitors of cysteine proteases (PCPIs) from
potato (Solanum tuberosum). A series of 20 kDa PCPIs was purified using a multi-step
chromatographical protocol, and two most abundant and effective isoinhibitors named
PCI 1-5 and PCI 3 were characterized in detail. They were screened against a broad
panel of model cysteine proteases and digestive cysteine proteases from herbivorous
insects. PCI 1-5 and PCI 3 exhibit different inhibitory specificity pattern and potency
up to the nanomolar range. Both isoinhibitors were crystallized and their spatial
structures were solved and refined at 1.5 Å (PCI 1-5) and 1.7 Å (PCI 3) resolutions.
A position of reactive sites against cysteine proteases on the conserved β-trefoil
fold scaffold was proposed. The work provides the first analysis of PCPIs with respect
to the structure-function relationships and evolution within the Kunitz-type inhibitor
family.
PB-019
Role of the ABCC2 transporter in the mode of action of the Bacillus thuringiensis
Cry1Ac toxin in the Diamond Back Moth Plutella xylostella
Josué Ocelotl1, Jorge Sánchez2, Raquel Arroyo1, Isabel Gómez1, Gopalan Unnithan2,
Bruce Tabashnik2, Alejandra Bravo1, Mario Soberón1
1Instituto de Biotecnología, Universidad Nacional Autónoma de México, 2Department
of Entomology, University of Arizona
Due to being environmentally friendly and highly specific to their target insect,
Bacillus thuringiensis three domain-Cry toxins (Bt toxins) are widely used as an alternative
to chemical insecticides in formulations or in transgenic expression. To exert their
toxic effect the protein crystals that contain this pore forming toxins must be ingested
by susceptible insect larvae. Once in the midgut, the crystal is solubilized and activated
by gut proteases. The protease resistant fragment, composed of three domains is able
to interact with different insect proteins located in the apical membrane of the gut
epithelium, putative Cry-toxins binding proteins include cadherin (CADR), aminopeptidase-N
(APN), and alkaline phosphatase (ALP). Current model for the mode of action involves
a low affinity interaction of Cry1A toxins with the highly abundant GPI-anchored-receptors,
ALP and APN which concentrates the toxin close to the microvilli membrane, here the
toxin binds in a high affinity interaction to the cadherin receptor, this event promotes
the proteolytic cleavage of the N-terminal end including helix α-1 triggering the
formation of an oligomer structure. The binding of oligomeric Cry1A structure to ALP
and APN facilitates insertion of this pre-pore into the membrane causing pore-formation
and cell lysis. It is known that mutations resulting in diminished or lack of expression
of Cry-toxin binding proteins result in high levels of resistance of the insect pests.
In recent years, a novel resistance mechanism involving an ABC transporter (ABCC2)
has been reported in four lepidopteran insects, Helothis virescens YEE and YHD3 strains,
Plutella xylostella NO-QA strain, Bombyx mori C2 strain and Trichoplusia ni GlenBtR
strain. Although some studies have suggested that the ABCC2 transporter could be a
receptor for Bt toxins in this insect pests, the precise role of this membrane protein
in the mode of action of Cry toxins remains unclear. Interestingly insects with mutations
in the ABCC2 transporter or cadherin show high resistance levels to Cry1Ab or Cry1Ac
toxins but are susceptible to mutant toxins named Cry1AbMod or Cry1AC Mod, in which
the N-terminal end of the toxin including helix-α1was deleted, these proteins do not
require the presence of cadherin to form oligomeric structures. We analyzed and compared
the binding interactions of Cry1Ac and Cry1AcMod toxins with brush border membranes
from susceptible and resistant populations of P. xylostella and P. gossypiella, which
display different Cry-toxin resistance mechanisms, ABCC2 or cadherin. Our data shows
that ABCC2 and cadherin are involved in Cry1Ac toxin oligomerization in P. xylostella
and P. gossypiella respectively showing that these receptor molecules fulfill the
same role in the mode of action of Cry1Ac in different insect species.
PB-020
Metabolic alkene labeling and in vitro detection of histone acylation via the aqueous
oxidative Heck reaction
Maria-Eleni Ourailidou1, Paul Dockerty1, Martin Witte1, Gerrit J. Poelarends1, Frank
J. Dekker1,
1University of Groningen
The detection of protein lysine acylations remains a challenge due to lack of specific
antibodies for acylations with various chain lengths. This problem can be addressed
by metabolic labeling techniques using carboxylates with reactive functionalities.
Subsequent chemoselective reactions with a complementary moiety connected to a detection
tag enable the visualization and quantification of the protein lysine acylome. In
this study, we present EDTA-Pd(II) as a novel catalyst for the oxidative Heck reaction
on protein-bound alkenes, which allows employment of fully aqueous reaction conditions.
We used this reaction to monitor histone lysine acylation in vitro after metabolic
incorporation of olefinic carboxylates as chemical reporters.
PB-021
“Study of Bacillus thuringiensis Cry1Ab and Cry1Ac protoxins interaction with cadherin-like
receptor from Manduca sexta”
Arlen Peña-Cardeña1, Alejandra Bravo1, Mario Soberón1, Isabel Gómez1,
1Instituto de Biotecnología, Universidad Nacional Autónoma de México
The Gram-positive bacterium Bacillus thuringiensis (Bt) produces insecticidal crystal
proteins (Cry toxins) to control insect pests. Cry toxins are recognized as pore forming
toxins that kill larval epithelium midgut cells by causing an osmotic shock leading
to cell lysis. To induce the pore formation of Cry toxins, the parasporal crystals
have to be ingested by susceptible larva, solubilized by the pH conditions of the
insect gut, and activated by midgut proteases to yield the resistant core of the activated
toxin. In the case of Cry toxins that are active against lepidopteran insects, it
has been shown that Cry1A toxins undergo a sequential binding mechanism with glycosyl-phosphatidyl-inositol
anchored proteins such as alkaline phosphatase (ALP) or aminopeptidase-N (APN) and
cadherin-like protein resulting in the formation of a pre-pore oligomeric structure
that is proficient in membrane insertion and pore formation. Receptor recognition
by Cry toxins has been recognized as a key step of Cry toxicity that is fundamental
for insect specificity. Previously we reported that Cry1Ab protoxin or activated toxin
bind cadherin-like receptor with similar affinities and two different pre-pores are
produced depending on which of these molecules interacts with cadherin in the presence
of insect midgut proteases. Here we test the interaction with a second protoxin the
Cry1Ac that share 85% of identity with Cry1Ab and both have similar toxicity against
Manduca sexta larva. However, we observe that Cry1Ac protoxin has no interaction with
cadherin-like receptor as Cry1Ab, our results suggest that another protein may act
as receptor for Cry1Ac protoxin.
PB-022
Proton solvation in protic and aprotic solvents
Emanuele Rossini1, Ernst-Walter Knapp1
1Institute of Chemistry and Biochemistry, Freie Universität Berlin
Protonation pattern influence actively properties of molecules and play an essential
role in biochemical mechanisms. For an accurate determination of the protonation equilibria,
the absolute proton solvation free energy needs to be known. The determination of
this energy represents one of the most challenging problems in physical chemistry.
This is particularly difficult for protons solvated in water, where the solvation
is dynamically performed by different water clusters and the proton is not attached
to a single solvent molecule. The proton solvation is notably important in order to
quantify mechanisms of proton transfer and such processes have been investigated for
a long time based on different approaches, often leading to contradictory conclusions.
A rigorous and accurate protocol for computing proton solvation in solvents of different
nature is of prime importance for applied (pharmaceutical and material science) and
fundamental sciences. In this study, proton affinities, electrostatic energies of
solvation and pKa values of a reference set of organic molecules are computed in protic
and aprotic solvents. Proportional to the free energy of proton dissociation, the
pKa value calculation is therefore strongly dependent on the free energy of proton
solvation. Such energy is then determined in acetonitrile (ACN), methanol (MET), water
and dimethyl sulfoxide (DMSO) in order to obtain the best possible match between measured
and computed pKa values. The computation of these values is based on a combination
of quantum chemical (QC) and electrostatic approaches by using a thermodynamic cycle
connecting gas-phase and solvent-phase of proton dissociation. The computed proton
solvation energies in ACN, MET, water and DMSO of the present study are very precise
(RMSD much lower than 1 pH value). They will be a basis for better understanding of
proton solvation and help to predict pKa values of organic compounds in different
solvents more precise.
PB-023
Biochemical characterization of two evolutionary distant ten-eleven translocation
enzymes and their utility in 5-methylcytosine sequencing in the genomes at single-base
resolution
Lana Saleh1, Esta Tamanaha, June Pais1, Romualdas Vaisvila1, Nan Dai1, Shengxi Guan1,
Ivan Correa1, Christopher Noren1, Richard Roberts1, Yu Zheng1
1New England biolabs
The ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC)
on DNA to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. Here,
we examine the in vitro biochemical activity of two evolutionary distant TETs, mTET1
from mouse and NgTET1 from the single-celled protist Naegleria gruberi. We show that
both of these enzymes are 5-methylpyrimidine oxygenases with activity on both 5mC
(major activity) and thymidine (T) (minor activity) and preference to 5mCpG and TpG
dinucleotide sites. Intriguingly, NgTET1 displays higher T oxidation activity in vitro
than mTET1 supporting a closer evolutionary relationship between NgTET1 and the base
J binding proteins from trypanosomes. In fact, unprecedented evidence for the formation
of two new bases, 5-formyluridine and 5-carboxyuridine, is shown for NgTET1 activity
in vitro. Mutagenesis studies performed in NgTET1 reveal a delicate balance between
choice of 5mC or T as the preferred substrate. Steady-state kinetic analysis show
that both mTET1 and NgTET1 are distributive in their oxidative chemistry with each
oxidized species released from the enzyme upon formation. Furthermore, both enzymes
are physically distributive in the recognition of their substrates on template DNA
with no apparent preference to a specific 5mC oxidation intermediate. These data indicate
a role for the TET enzymes in the maintenance of the three oxidized forms of 5mC and
suggest that these bases are not simply intermediates in a methylation cycle but represent
additional epigenetic states in genomic DNA with regulatory functions to be explored.
Finally, we demonstrate the utility of both enzymes in 5mC sequencing technologies
and present examples for the mapping of 5mC in different genomes at base resolution.
PB-024
Contribution of Connexin37 Gene Polymorphism (C1019T) in the Incidence of Acute Myocardial
Infarction in the Egyptians
Fadwa El Tahry1, Ingy Hashad1, Mohamed Farouk1, Mohamed Gad1
1German University In Cairo (GUC)
Contribution of Connexin37 Gene Polymorphism (C1019T) in the Incidence of Acute Myocardial
Infarction in the Egyptians Fadwa A. El Tahry1, Ingy M. Hashad1, Mohamed F. Abdel
Rahman1, Mohamed Z. Gad1 Clinical Biochemistry unit1 Faculty of pharmacy and Biotechnology,
German University in Cairo, Egypt Background: Cardiovascular diseases are the leading
cause of death worldwide and in Egypt. Acute myocardial infarction (AMI), the main
outcome of ischemia, is caused mainly by atherosclerosis. Connexin 37 (Cx37) protein
plays a protective role by decreasing the monocyte adhesion, therefore delaying the
initiation of atherosclerosis and as a result the incidence of AMI. Gap junctions
are protein structures present between cells that help in the electrical and metabolic
coupling of cells. Their building subunits are connexins. To date 21 connexins (Cxs)
have been identified in humans. CX37 is one of the Cxs forming the gap junction protein
family. It is expressed mainly on vascular endothelial cells and monocytes. Several
studies reported a single nucleotide polymorphism (SNP) C1019T in CX37 gene as a prognostic
marker for atherosclerosis. Aim of work: To investigate the contribution of C1019T
gene polymorphism of CX37 in the predisposition of AMI in the Egyptians. Subjects
and Methods: The study consisted of 114 AMI patients and 100 controls. Blood was taken
after taking a written consent from all subjects and the approval of the ethical committees
of EL Demrdash hospital and the German university in Cairo. DNA was extracted from
the whole blood using Thermo-scientific DNA extraction kit. The frequency of the genotypes
(CC, TT and CT) was determined by (PCR-RFLP) using Drd I restriction enzyme. Results:
The genotype distribution of CX37 gene in AMI patients was (CC 15.78%, TT 11.40%,
CT 72.80%) while in controls (CC 27%, TT 18%, CT 55%) (p =0.516). On the other hand,
the alleles frequencies of AMI patients were (C allele=52.19%, T allele= 47.80%) and
in control subjects (C allele= 54.5%, T allele=45.5%(p=0.697). Conclusion: The CX
37 gene polymorphism (C1019T) is not associated with the incidence of AMI in the Egyptians.
PB-025
Delineating toxin:lipid:ion channel interactions for rationally sodium channel inhibitors
design
Christina Schroeder1, Sónia Henriques2, Mehdi Mobli2, Stephanie Chaousis1, Phillip
Walsh1, Panumart Thongyoo1, David Craik1
1Institute for Molecular Bioscience, The University of Queensland, 2Centre for Advanced
Imaging, The University of Queensland
In recent years, certain voltage-gated sodium channels (NaV) subtypes have emerged
as validated chronic pain targets with loss-of-function and gain-of-function mutations
in both NaV1.7 and NaV1.8 subtypes leading to an inability to perceive pain and painful
neuropathies, respectively. However, as NaV ion channels are intimately involved in
almost all aspects of physiology, only the most selective inhibitors would be suitable
as drug leads. Disulfide-rich venom derived mini-proteins from cone snails and spiders
are being actively pursued as novel therapeutics for pain, because of their high selectivity
and potency at human ion channels, including sodium channels (NaV). Two main strategies
of inhibition have been identified; blocking the pore and interacting with the voltage-sensor
domains (VSD) surrounding the pore. The ion-conducting pore is highly conserved between
all sodium channel subtypes whereas the voltage-sensor domain binding sites are less
conserved. Therefore, inhibition of a specific NaV isoform is more achievable using
inhibitors that modulate VSDs than with pore blockers. Gating modifier toxins from
spider and cone snail venom inhibit NaV1.7 and NaV1.8 by interacting with the VSD.
They appear to reach their target by partitioning into the lipid membrane surrounding
the ion channel, thus enabling access to the VSD. Toxin pharmacology may therefore
not only be driven by the peptide-ion channel interactions, but also including the
lipids surrounding the channel protein, a feature that is very much under explored.
It is therefore apparent that peptide-lipid interactions in combination with peptide-channel
interactions need to be considered when designing potent inhibitors. Using a range
of biophysical techniques, including surface plasmon resonance and nuclear magnetic
resonance, we are studying the interactions underpinning the mechanism of action between
toxins and membranes and toxins and ion channels. Initial results show that the lipid
composition surrounding ion channels play a major role in terms of toxin:lipid interaction
and that these interactions can be used in combination with traditional structure-activity
relationship studies to design selective and potent NaV inhibitors, which will be
discussed. We believe that our studies will ultimately delineate what drives toxin
pharmacology and NaV subtype selectivity and will lead to improve rationally engineering
of novel therapeutics for the treatment of pain.
PB-026
Micelles promote Aß42 assembly into pore-forming oligomers
Montserrat Serra-Batiste1, Mariam Bayoumi2, Margarida Gairí3, Martí Ninot-Pedrosa1,
Giovanni Maglia2, Natàlia Carulla1
1Institute for Research in Biomedicine (IRB Barcelona), 2Biochemistry, Molecular and
Structural Biology Section, University of Leuven, 3NMR Facility, Scientific and Technological
Centers, University of Barcelona
The formation of amyloid-β peptide (Aβ) oligomers at the cellular membrane is considered
to be a crucial process underlying neurotoxicity in Alzheimeŕs disease (AD). 1-2 Therefore,
it is important to understand how oligomers form within a membrane environment. Using
solution nuclear magnetic resonance (NMR) spectroscopy, combined with size exclusion
chromatography (SEC), we have studied the two major Aβ variants— Aβ40 and Aβ42, the
latter having a more prominent role in AD than the former—under carefully selected
micelle conditions intended to mimic a membrane environment. Our results indicate
that after an incubation period, Aβ42, but not Aβ40, assembles into oligomers with
specific structural properties, which we have named Stabilized Micelle Oligomers (SMOs).
SMO complexes incorporate into lipid bilayers as well-defined pores, a feature linked
to neurotoxicity. These results have important implications in the AD field as they
provide a new perspective on how Aβ oligomers cause neurotoxicity. Indeed, our findings
constitute a first step towards the establishment of a new therapeutic target for
AD. 3
1. S. M. Butterfield, H. A. Lashuel (2010) Amyloidogenic protein-membrane interactions:
mechanistic insight from model systems. Angew Chem Int Ed Engl 49(33):5628-5654. 2.
S. A. Kotler, P. Walsh, J. R. Brender, A. Ramamoorthy (2014) Differences between amyloid-beta
aggregation in solution and on the membrane: insights into elucidation of the mechanistic
details of Alzheimer’s disease. Chem Soc Rev. 3. M. Serra-Batiste, M. Bayoumi, M.
Gairí, M. Ninot-Pedrosa, G. Maglia, N. Carulla (2015) Micelles promote Aβ2 assembly
into pore-forming oligomers. Under minor revisions in Proc Natl Acad Sci USA.
PB-027
Molecular dynamics study on the key catalytic intermediates of threonine synthase
Mitsuo Shoji1, Yuzuru Ujiie1, Ryuhei Harada1, Megumi Kayanuma1, Yasuteru Shigeta1,
Takeshi Murakawa2, Hideyuki Hayashi2
1Univeristy of Tsukuba, 2Osaka Medical College
Threonine Synthase (ThrS) catalyzes a formation of L-threonine from O-phospho-L-homoserine.
The series of reactions catalyzed by ThrS encompasses many regiospecific and stereospecific
steps, which are controlled by the enzyme protein, However, the precise mechanism
of the reaction control (product-assisted catalysis) is not fully elucidated. In this
study, molecular dynamics (MD) simulations of ThrS were performed with the thermodynamics
integration approach, and the accurate free energy differences between the key intermediates
were evaluated by changing the phosphate ion to a sulfate ion. It is already known
experimentally that the phosphate ion is one of the products of the enzyme reaction
and plays an important role in the catalytic reaction. The free energy differences
between the ions were well reproduced theoretically for the key intermediate states.
By performing additional 100ns MDs, we analyzed the substrate conformations. We found
that the substrate conformations are changed by the reaction-controlling ions. This
finding suggests that the controlling of the substrate conformation will be an important
molecular mechanism for the product-assisted catalysis.
PB-028
Agrobacterium tumefaciens employs two distinct ClpS adaptors to modulate the N-end
rule degradation pathway
Benjamin J. Stein1, Robert A. Grant1, Robert T. Sauer1, Tania A. Baker1,2
1Department of Biology, Massachusetts Institute of Technology, 2Howard Hughes Medical
Institute, Massachusetts Institute of Technology
The N-end rule is a widely conserved proteolytic pathway, in which the N-terminal
amino acid of a protein determines its in vivo stability. In E. coli and C. crescentus,
the ClpS adaptor protein recognizes destabilizing N-termini and delivers them to the
ClpAP AAA+ protease for degradation. Unlike the majority of proteobacteria, most α-proteobacteria
contain two paralogs of ClpS, ClpS1 and ClpS2. Here, we investigate the binding specificity
of the ClpS1 and ClpS2 proteins from A. tumefaciens. We demonstrate that both ClpS1
and ClpS2 deliver N-end rule substrates to ClpA, but ClpS2 has a narrower binding
specificity than ClpS1. Importantly, crystal structures of ClpS2 reveal conformational
changes in the substrate-binding pocket that are critical for N-end rule recognition.
Moreover, we find evidence that ClpS1 and ClpS2 are differentially expressed in A.
tumefaciens. We conclude that A. tumefaciens contains two ClpS proteins with differing
N-terminal binding specificities, allowing fine-tuning of N-end rule recognition at
the level of adaptor proteins.
PB-029
Interactions between U24 from HHV-6A and 7 and Nedd4 or Smurf2 WW domains
Yurou Sang1, Rui Zhang1, Walter R.P. Scott1, A. Louise Creagh2, Charles A. Haynes2,
Suzana K. Straus1
1Department of Chemistry, University of British Columbia, 2036 Main Mall, 2Michael
Smith Labs, University of British Columbia
U24 is a protein found in both Human Herpes Virus type 6A (HHV-6A) and type 7 (HHV-7),
with an N-terminus which is rich in prolines (PPxY motif in both HHV-6A and 7; PxxP
motif in HHV-6A). Previous work tested the hypothesis that U24 may be implicated in
multiple sclerosis (MS), because of a shared seven residue sequence identity between
U24 and myelin basic protein (MBP), a key protein in the progression of MS. Our study
showed however that the binding between U24 (via the PxxP motif) and its interaction
partner Fyn-SH3 was weak [1]. The current study examines the interaction of U24 (via
the PPxY motif) with WW domains, small protein-protein interaction domains of approximately
40 residues that derive their name from two highly conserved tryptophan residues,
usually spaced 22-23 amino-acids apart. The interaction between U24 and WW domains
is deemed to be important for endocytic recycling of T-cell receptors [2]. Binding
affinities of a number of U24-WW pairs were determined in order to identify whether
a specific binding partner exists. Data from ITC, NMR and molecular dynamics simulations
will be presented and discussed in light of the function of U24 in disease, with a
particular focus on MS.
[1]- Y. Sang, A.R. Tait, W.R.P. Scott, A.L. Creagh, P. Kumar, C.A. Haynes, S.K. Straus,
Biochemistry, 53(38):6092-102 (2014).
[2]- B.M. Sullivan and L. Coscoy, J. Virol., 84(3):1265-75 (2010).
PB-030
Ebola Virus Surface Glycoprotein GP2 Forms a Hydrophobic Fist to Enter Cells by Membrane
Fusion
Jinwoo Lee1, Sonia Gregory1, Lukas Tamm1
1University of Virginia
Ebola Virus (EboV) is a filamentous membrane-enveloped virus that enters cells by
macropinocytosis and pH-triggered membrane fusion. The disulfide-stabilized fusion
loop of Ebola GP2 is extended on the surface of circulating virus, but forms a compact
hydrophobic “fist” in the Nieman-Pick late endosomal compartment. The pH-induced conformational
change of the EboV fusion loop enables the virus to escape from the endosome into
the cytoplasm where it replicates causing severe hemorrhagic fever in infected individuals.
We determined the structure of the EboV fusion loop and several fusion-compromised
mutants by NMR. The results show that the fist is stabilized by a hydrophobic triad
consisting of a leucine, an isoleucine and a phenyalanine residue. The fist interacts
with residues in the membrane-proximal and transmembrane domains of GP2, the structures
of which we also determined by NMR, to catalyze membrane fusion and virus entry into
the cytoplasm. These protein interactions that involve a dramatic refolding of the
protein at the membrane surface provide potential targets for drug discovery and new
strategies for vaccine development. Supported by NIH grant R01 AI30557.
PB-031
Zero-Length Crosslinking of the ß Subunits of the Phosphorylase Kinase Complex by
Periodate
Jackie Thompson1, Owen Nadeau1, Gerald Carlson1
1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center
Phosphorylase kinase (PhK) is a hexadecameric enzyme complex that regulates glycogen
catabolism. The complex is composed of four copies of four subunits: α, β, γ and δ.
The α, β and δ; subunits regulate the catalytic γ; subunit through quaternary constraints
that inhibit its activity. Upon binding allosteric effectors or the covalent modification
of the regulatory subunits, the γ subunit is activated. Despite nearly six decades
of investigation, the subunit-interactions responsible for the inhibition and activation
of the γ subunit are still largely unknown. Due to PhK’s large size (1.3 megadaltons),
studying its structure is challenging, but chemical crosslinking has proved a valuable
tool to study interactions among its α, β, γ; and δ; subunits before and after activation
by phosphorylation. Here we report selective, zero-length crosslinking of the regulatory
β subunits of PhK by the general oxidizing agent periodate, even at concentrations
as low as µM. SDS-PAGE, mass spectrometry, Western blot analysis, and size-exclusion
chromatography were used to characterize crosslinked PhK and to identify the products
formed. Oxidation of non-activated PhK at pH 6.8 with periodate produced a β-dimer,
making periodate the most effective crosslinking agent to produce this homodimer.
When periodate crosslinking of non-activated PhK was carried out in the presence of
a synthetic peptide corresponding to the N-terminal 22 residues of β (Nβ peptide)
known to compete with its counterpart region in the native enzyme and disrupt interactions
between the β and γ subunits, there was an increased amount of β-dimer formation.
It should be noted that this Nβ peptide contains the autophosphorylatable Ser-11 associated
with PhK activation, and phosphorylated Nβ; peptide was considerably less effective
in promoting β-dimer formation than non-phosphorylated peptide. These results suggest
a role for Ser-11 autophosphorylation in mediating homodimeric β subunit interactions
within the PhK complex, and augment previous studies on the activation of PhK by phosphorylation
in which changes at the N-terminus of β are critical in the activation of the catalytic
γ subunit. Summing these results leads to a new model of activation. In this model,
in the inactive state, the nonphosphorylated N-terminus of β interacts directly or
indirectly with the regulatory C-terminal domain of the γ subunit, inhibiting catalytic
activity. Upon phosphorylation of the N-terminus of β, three important events occur:
1) the interaction between β and γ is disrupted, 2) the β subunits of the holoenzyme
self-associate, and 3) the catalytic domain is activated. Thus, we envision that the
N-terminus of β acts as an allosteric switch, with activation triggered by phosphorylation
of this region, causing disruption of its previously inhibiting interactions with
γ and promotion of β β dimerization to stabilize the activated conformation of γ.
The research was supported financially by the University of Kansas Medical Center
Biomedical Research Training Program and NIH Grant DK32953.
PB-032
hSSB1 is involved in the cellular response to oxidative DNA damage
Christine Touma1, Nicolas Paquet2, Derek J. Richard2, Roland Gamsjaeger1,3, Liza Cubeddu1,3
1School of Science and Health, University of Western Sydney, 2Queensland University
of Te chnology, 3School of Molecular Bioscience, University of Sydney
Cellular DNA is subject to oxidative damage in the presence of reactive oxygen species.
The 7,8-dihydro-8-oxoguanine (8-oxoG) adduct is the most common form of oxidative
damage and results in G:C to T:A transversions; these lesions are normally processed
by the Base Excision Repair (BER) pathway. Single-stranded binding (SSB) proteins
of the oligonucleotide binding domain family are heavily involved in DNA repair processes,
which involve the detection of DNA damage and recruitment of repair proteins to the
site of damage. Using immunofluorescence we demonstrate that hSSB1 (a novel human
SSB) levels increase in response to oxidative damage (H202). Cells depleted of hSSB1
are hypersensitive to oxidative damage and are also unable to efficiently remove 8-oxoG
adducts. We show that hSSB1 forms dimers and tetramers under oxidative conditions
and that this oligomerisation is likely mediated by inter-domain disulfide bond formation.
Furthermore, using Surface Plasmon Resonance, we also show that oxidised hSSB1 binds
to 8-oxo-G damaged ssDNA with higher affinity than non-damaged ssDNA, indicating a
direct role for oxidised hSSB1 in the recognition of 8-oxo-G lesions. As oxidative
stress is associated with aging, cancer and Alzheimer’s disease, understanding the
molecular mechanisms of how cells repair oxidative DNA damage will be crucial in the
development of potential therapeutic treatments.
PB-033
Virtual Screening for Novel Inhibitors of Acetoacetyl-CoA Reductase of Burkholderia
pseudomallei
Luis Valencia1,2, Josh Beckham, Oscar Villarreal, Jon Robertus
1University of Texas at Austin, 2Freshman Research Initiative
Burkholderia pseudomallei is a gram-negative bacteria that causes the disease melioidosis,
a potentially chronic and aggressive infection with a mortality rate of up to 90%
and is listed as a category B critical biological agent by the National Institute
of Allergy and Infectious Diseases (NIAID). The glyoxylate metabolism pathway of B.
pseudomallei carries out the metabolism of fatty acids – a function responsible for
the virulent ability of B. pseudomallei to survive after being engulfed by the host’s
macrophages. Virtual screening on the structure of acetoacetyl-CoA reductase, a protein
suggested to be essential in the glyoxylate pathway of B. pseudomallei, was conducted
using GOLD molecular docking program to identify potentially novel inhibitor ligands
from large virtual chemical libraries. The gene sequence of the acetoacetyl-CoA was
assembled through overlap PCR and inserted into the expression vector pNIC-Bsa4 using
ligation independent cloning. The protein was then expressed using IPTG induction
of the T7 polymerase Lac operon system in BL21(DE3) cells, purified using His-tag
Ni- NTA affinity chromatography, and characterized using SDS-PAGE. Enzymatic activity
was confirmed by using a spectrophotometric enzyme assay measuring the absorbance
of NADPH at 340nm during the reduction of acetoacetyl-CoA into (R)−3-Hydroxy-butanoyl-CoA.
From the virtual screening of 30,000 ligands of the Chembridge Diversity Library against
the acetoacetyl-CoA reductase structure the highest scoring ligands were selected
and ordered for inhibition assay experiments. Current research objectives involve
identification of novel inhibitors from inhibition assays, determination of enzyme
kinetics, and the development of an acetoacetyl-CoA reductase structure with ligands
or inhibitors bound in the active site.
PB-034
Use of Principal Component Analysis and Molecular Docking to Identify Novel Selective
Plasmepsin II Non-Competitive Inhibitors with Antimalarial Activity
Pedro Alberto Valiente Flores1, Maarten G Wolf2, Yasel Guerra3, Isel Pascual1, Isabelle
Florent4, Enrique Rudiño3, Pedro Geraldo Pascutti5, Tirso Pons6, Gerrit Groenhof2
1Center of Protein Studies, Faculty of Biology, University of Havana., 2Max Planck
Institute of Biophysical Chemistry, 3Biotechnology Institute. UNAM, 4CNRS-MNHN, 5Biophysics
Institute. Federal University of Rio de Janeiro, 6Spanish National Cancer Research
Centre
Plasmepsin II (PlmII) is an aspartic protease involved in the initial steps of the
hemoglobin degradation pathway, a critical stage in the Plasmodium falciparum life
cycle during human infection. However, most of the PlmII inhibitors obtained through
structure-based ligand design have generally shown a low selectivity towards the human
related protease Cathepsin D (hCatD), which is a notable drawback to their use as
antimalarial drugs. Here, we presented a novel in silico approach based on the combined
use of principal component analysis and molecular docking to identify non-competitive
selective inhibitors of PlmII. We searched unique conformational states of PlmII that
can not be adopted by the human aspartic proteases: Cathepsin D, Renin and Pepsin
by comparing the conformational subspaces sampled by these proteins along molecular
dynamic simulations of 1.2 µs. Specific conformations along the flap opening-siding
mode of PlmII that can not be sampled by the human counterparts were identified. The
specific conformations were used to perform virtual screening experiments and proposed
putative PlmII selective-inhibitors. The hCatD was also targeted to exclude non-selective
compounds. The first five ranked inhibitors, with inhibition constants (Ki) values
in the µM-nM range, target a cryptic flap interior pocket formed by the residues M75,
V82, V105, T108, and Y115 which is only exposed in the PlmII specific conformations.
The inhibition assays showed that the inhibitors bind better PlmII than hCatD in a
range from 70 to 100-fold of their Ki values. The kinetic characterization showed
a non-competitive inhibition of PlmII for all the compounds. Molecular docking calculations
suggest that these compounds probably target the other three Plms expressed in the
digestive vacuole of Pf. Notably two of them (SPB07935 and HTS07519) inhibited red
blood cell cultures infected with the Pf cloroquine resistant strain FcB1 with an
IC50 value in the µM range.
PB-035
Ain’t gold all that glitters: missing gold atoms in the structure of lysozyme crystals
used to co-crystallize gold nanoparticles
Antonello Melrino1, Irene Russo Krauss2, Marco Caterino1, Alessandro Vergara1
1Dept. Chemical Sciences, University of Naples Federico II, 2Institute of Biostructures
and Bioimaging, CNR
Wei et al. (2011) reported the growth of gold nanoparticles within protein single
crystals of hen egg white lysozyme (HEWL) [1]. We tried to reproduce the experiments
performed by these authors obtaining red, well-diffracting crystals of HEWL after
1 month of soaking in the presence of the gold-nanoparticle precursor ClAuS(CH2CH2OH)2.
However, when refining our crystal structures we found no gold atoms.[2] This finding
prompted us to analyze the models by Wei et al. deposited in the Protein Data Bank
(codes 3P4Z, 3P64, 3P65, 3P66, 3P68), where nine different gold atoms are present
(four isolated gold atoms and a 5-atom cluster). For eight out of the nine gold atoms
found in the structures deposited by Wei et al., we believe that the authors’ interpretation
is questionable. [2] Ultimately, three out of five crystal structures solved by Wei
et al. likely correspond to HEWL with only one Au+ ion bound to His15, as previously
reported in crystal structures of adduct between HEWL and gold-based drugs [3-4].
The last two crystal structures are gold-free. We consider the ability of gold nanoparticles
to grow within protein single crystals a stimulating result that has interesting implications.
However, the structural analysis by Wei et al cannot be used to unveil protein-gold
nanoparticle interactions because no gold atom is unambiguously found in the reported
HEWL structures, apart from one ion bound to His15 in the first three structures.
This means that structural data on biomolecule-directed gold clusters is still lacking
and that the molecular basis of protein-gold nanoparticle recognition requires further
investigation.
References:
1. Wei, H. et al. Nature Nanotech. 6, 93–7 (2011).
2. Merlino, A. et al. Nature Nanotech. 10 (4), 285 (2015).
3. Messori, L. et al. Chem. Commun. 49 (86), 10100-2 (2013).
4. Russo Krauss et al. Dalton Trans 43 (46), 17483-8 (2014). Sponsored by the member
Prof. Filomena Sica
PB-036
Finding a novel treatment for the biological weapon threat of epidemic typhus by targeting
ß-ketoacyl-ACP-reductase in Rickettsia prowazekii
Oscar Villarreal1, Josh Beckham1, Jon Robertus1
1University of Texas at Austin, Department of Molecular Biosciences
Epidemic typhus, which is caused by the bacterial pathogen Rickettsia prowazekii,
is a menacing disease world wide that the NIH lists as one of America’s greatest biological
weapons threats. This research seeks to find novel inhibitors of β-ketoacyl-ACP-reductase
(FabG), an enzyme that catalyzes one of the reactions in the fatty acid synthesis
type II system in bacteria. This pathway is essential for survival in bacteria. The
FabG enzyme uses NADPH as a substrate, which facilitates the binding of the second
substrate, acetoacetyl-ACP into the active site. The acetoacetyl-ACP is subsequently
reduced into β-hydroxyacyl-ACP. The coding DNA sequence for the RpFabG protein was
cloned into a pNIC vector and transformed into E.coli BL21(DE3), then the protein
was expressed and purified using metal affinity and size exclusion chromatography
methods. High throughput molecular docking software (GOLD) was used to screen a commercial
library of ligands against the acetoacetyl-ACP region of the active site. The ligands
with the best GOLD scores were selected to be tested in vitro. Spectrophotometric
enzyme inhibition assays were performed to determine whether the drugs could inhibit
RpFabG activity. Chlorogenic acid, a previously known inhibitor of homologous FabGs,
was tested along with the other potential drugs, and was determined to have moderate
inhibitory effects on RpFabG. Loop modeling using ICM software was performed in order
to create a prediction of the complete RpFabG structure, including the disordered
loops that are not a part of the 3F9I PDB structure. Co-crystallization of RpFabG
with both substrates was carried out in order to obtain a structure, but only non-diffracting
crystals resulted. Further inhibition assays and crystallography trials are being
performed in order to continue the search for a novel inhibitor of RpFabG and ultimately
a treatment for epidemic typhus.
PB-037
Diazotransfer reagents to selectively functionalize a protein of interest with azido
groups
Martin Witte1, Jonas Lohse1, Remko Welker1
1Stratingh Institute for Chemistry, University of Groningen
The incorporation of a non-natural functional group onto proteins facilitates studying
their role. It also led to the development of protein variants which can be activated
(photo)chemically inside cells and it has become an important means to synthesize
biopharmaceuticals. The functional group is generally incorporated using auxotrophic
strains, amber suppression technology, enzymatic labelling or ligand-tethered labelling.1
These techniques, except for the latter, all require genetic modification and overexpression
of the target protein, which can lead to artefacts. Our aim was to develop reagents
that enable direct functionalisation of a target protein with a ligation handle in
a complex mixture. As requirement, we set that the handle should not only facilitate
further modification, but that it should also serve as a potential chemical turn-on/turn-off
switch. Conversion of an amine into an azido group fulfils these requirements. It
can be modified using bioorthogonal reactions, such as the copper-catalyzed click
reaction and the Staudinger ligation. Moreover, modification of essential amine groups
in the protein of interest may inhibit protein activity. Reduction of the azido group
will give the unmodified target protein and restore the activity, and as such the
azido group may also serve as a chemical turn-on/turn-off switch. Van Hest and coworkers
revealed that azido groups can be introduced onto purified recombinant proteins using
a diazotransfer reaction,2 but the described method cannot be used to selectively
modify a proteins of interest within a complex mixture. We reasoned that this issue
could be addressed by tethering the diazotransfer reagent to a ligand that binds to
the protein of interest. As a proof of concept, we synthesized novel reagents to selectively
functionalize the model proteins streptavidin and carbonic anhydrase II. The diazotransfer
reagents react selectively with their corresponding target protein in mixture of proteins
and when spiked in a cell lysate. Rapid labelling was observed in the presence of
Cu(II), but the diazotransfer reaction also occurred in the absence of copper, albeit
with reduced efficiency. To identify the site of modification, labelled proteins were
analyzed with mass-spectrometry. A single lysine residue is modified in the case of
streptavidin and tethering the reagents may thus enhance the site selectivity as well.
However, multiple lysine residues were modified in carbonic anhydrase II indicating
that careful design of the reagent is required to achieve site selective modification.
The experiments with streptavidin and carbonic anhydrase II revealed that targeted
diazotransfer reagents can be used to selectively modify a protein of interest, and
we are currently extending our approach to other proteins, such as the proteasome.
Figure 1.
Schematic representation of the approach
1 Takaoka, Y.; Ojida, A.; Hamachi, I. Angew. Chem. Int. Ed. 2013, 52, 4088.
2 Schoffelen, S.; van Eldijk, M. B.; Rooijakkers, B.; Raijmakers, R.; Heck, A. J.
R.; Van Hest, J. C. M. Chem. Sci. 2011, 2, 701.
PB-038
PENG: a neural gas-based approach for pharmacophore elucidation. method design, validation,
and virtual screening for novel ligands of lta4h
Sandra Kerstin Wittmann1, Daniel Moser1, Jan Sebastian Kramer1, René Blöcher1, Janosch
Achenbach3, Denys Pogoryelov2, Eugen Proschak1
1Institute of Pharmaceutical Chemistry, LiFF/OSF/ZAFES, Goethe-University, 2Institute
of Biochemistry, Goethe University, 3BASF SE
Leukotriene A4 hydrolase (LTA4H; EC 3.3.2.6) is a bifunctional zinc metalloprotease
which displays both epoxide hydrolase and aminopeptidase activity [1]. With a high
preference the leukotriene A4 hydrolase cleaves tripeptides at an arginyl bond on
NH2 position [3]. To screen for new inhibitors of the enzyme we used a growing neural
gas (GNG)-based approach for the extraction of the relevant features which we called
PENG (pharmacophore elucidation by neural gas). The results of the prospective virtual
screening have been validated using a fluorescence-based assay system. Therefore the
non-fluorescent substrate L-arginine-7-amido-4-methylcoumarin hydrochloride is used.
The LTA4H cleaves the arginyl bond what results in the fluorescent 7-amino-4-methylcoumarin.
Additionally, we could show that the PENG approach is able to predict the binding
mode of the ligand by X-ray crystallography.
References:
[1] J.Z. Haeggström, F. Kull et all., Leukotriene A4 Hydrolase., Prostaglandins &
other Lipid Mediators 68-69 (2002); 495-510.
[2] Y. Michael Shim, Mikell Paige. Leukotriene A4 Hydrolase – An envolving target.
Inflammatory Diseases - Immunopathology, Clinical and Pharmacological Bases, Dr Mahin
Khatami (Ed.), ISBN: 978-953-307-9118-0.
[3] Lars Orning, J.K. Gierse, and F. A. Fitzpatrick. The Bifunctional Enzyme Leukotriene-A,
Hydrolase Is an Arginine Aminopeptidase of High Efficiency and Specificity. J. Biol.
Chem. (1994); 269(15); 11269-73.
[4] J. Z. Haeggström and C. D. Funk. Lipoxygenase and Leukotriene Pathways: Biochemistry,
Biology, and Roles in Disease. Chem. Rev. (2011); 111; 5866 – 98.
[5] Robert J. Snelgrove et al. A critical role for LTA4H in limiting chronic pulmonary.
Science (2010) 330 (6000): 90-94
PB-039
Stabilization of aspergillus parasiticus cytosine deaminase by immobilization on calcium
alginate beads improved enzyme operational stability
Hassan Zanna1, Andrew Nok2, Sani Ibrahim2, Hauwa Inuwa2
1University of Maiduguri, 2Ahmadu Bello University
Abstract Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life
of 1.10 hrs at 37ºC, was stabilized by immobilization on calcium alginate beads. The
immobilized CD had pH and temperature optimum of 5 and 50ºC respectively. The immobilized
enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with
the apparent K’M values of 0.60 mM and 0.65 mM respectively, displaying activation
energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads
gave the highest yield of apparent enzymic activity of 51.60% of the original activity
and the enzymic activity was lost exponentially at 37ºC over twelve (12) hours with
half- life of 5.80 hrs. Hence, the operational stability of native CD can be improved
by immobilization on calcium alginate beads.
*Author for correspondence
PB-040
Ubiquitin-nanoparticle interactions by solution NMR spectroscopy
Serena Zanzoni1, Michael Assfalg1, Rajesh K Singh2, Marco Pedroni3, Adolfo Speghini3,
David Fushman2, Mariapina D’Onofrio1
1NMR Laboratory, Department of Biotechnology, University of Verona, 2Center For Biomolecular
Structure and Organization, Department of Chemistry and Biochemistry, University of
Maryland, 3Solid State Chemistry Laboratory, Department of Biotechnology, University
of Verona
The potential use of nanoparticles (NPs) in biomedical applications has attracted
considerable interest in the last years. NPs introduced in a biological environment
interact with a collection of biomolecules, including proteins. NPs associating with
proteins may determine changes in protein conformation, interfere with protein-protein
interactions, and affect signal communication pathways [1]. Therefore, the study of
NP-induced functional perturbations of proteins implicated in the regulation of key
biochemical pathways is particularly relevant. Ubiquitin (Ub) is a small cytosolic
protein playing a central role in numerous biological processes including protein
degradation, cell signaling, and DNA repair [2]. It can be predicted that its interaction
with NPs may affect cellular pathways. In this respect, we characterized, at atomic
level, the interactions of Ub with two different size and chemical composition NPs.
The first NP that was tested was fullerenol, a polyhydroxylated [60]fullerene NP.
These carbon based NPs have several potential biomedical applications, including their
use as drug carriers, antiviral drugs, enzyme inhibitors, contrast agents, antioxidants,
and photosensitizers [3]. To characterize the fullerenol-Ub interactions, site-resolved
chemical shift and intensity perturbations of Ub’s NMR signals, together with 15N
spin relaxation rate changes, were used [4]. The obtained data were consistent with
interactions involving fullerenol clusters adsorbing reversibly to monomeric Ub (and
dimeric Ub), and targeting specific binding epitopes, coincident with functional recognition
sites of Ub. Furthermore, we observed that fullerenol almost completely abolished
the formation of di-Ub and longer chains in vitro, suggesting that fullerenol NPs
may effectively interfere with protein-mediated functional communication, eliciting
cytotoxic effects. The second kind of examined NPs were SrF2 NPs. Considerable importance
in biomedical luminescence is the application of these NPs doped with rare lanthanide
ions, due to their ability to produce up-conversion emission [5]. NMR spectroscopy,
up-conversion luminescence measurements and isothermal titration calorimetry were
used to probe the Ub-SrF2 NP interactions. As in the case of fullerenol NPs, the analysis
of NMR data indicated the occurrence of a reversible equilibrium between free and
NP-bound protein forms. The identification of similar interaction epitopes suggests
a similar impact on functional biomolecular communication. Our findings support the
view that NPs may affect fundamental interaction patterns of Ub, with possible nanotoxic
consequences on cell homeostasis. On the other hand, the specific inhibition of critical
Ub interactions through competitive binding of NPs to polyUb chains could represent
a new potential opportunity for pharmacological intervention against cancer development.
Reference:
1. Saptarshi S.R. et al., J. Nanobiotechnology, 2013, 11, 26.
2. Aguilar R.C. et al., Curr. Opin. Cell Biol., 2003, 15, 184–190.
3. Bosi S. et al., Eur. J. Med. Chem., 2003, 38, 913–923.
4. Zanzoni S et al., Nanoscale, 2015, DOI: 10.1039/C5NR00539F 5. Dong N.N. et al.,
ACS Nano, 2011, 5, 8665–8671.
PB-041
Chemical-Genetic Dissection of Protein Kinase Functions
Chao Zhang1, Ying-Chu Chen1, Alvin Kung1
1Department of Chemistry, University of Southern California
Small molecules are extremely useful tools for elucidating protein functions in cells
due to their acute and tunable effects. However, they often have difficulty distinguishing
between target proteins that are highly homologous to each other. To address this
deficiency and achieve high specificity, we are developing molecules that can covalently
target a reactive feature in the target protein. The reactive feature, in the form
of a cysteine residue near a ligand-binding pocket, can be naturally present or engineered
via mutagenesis in the protein of interest. Using this chemical-genetic approach,
we successfully identified specific inhibitors for a single isoform of Eph receptor
tyrosine kinases. These isoform-selective Eph inhibitors allow us to evaluate the
role of individual Eph kinases in cells. In addition, we applied the chemical-genetic
approach to the study of the Raf serine/threonine kinases, a key player in the MAPK/ERK
pathway (also known as the Ras-Raf-MEK-ERK pathway) that plays an essential role in
the regulation of cell proliferation, differentiation and survival. Surprisingly,
the selective inhibitors of oncogenic BRAF generated in this manner were unable to
completely block the MAPK signaling in cells, which we attribute to the transactivation
of endogenous wide-type RAF in cells. These results suggest that a pan-RAF inhibitor
is required for suppression the MAPK signaling in cancer cells. These findings have
direct implications for the drug resistance observed in the clinic and the development
of second-generation melanoma therapies.
PB-042
Selective modification of proteins and peptides by ruthenium porphyrin-catalyzed carbene
transfer reaction
Chi-Ming Ho1, Jun-Long Zhang1, Cong-Ying Zhou1, On-Yee Chan1, Jessie Jing Yan1, Fu-Yi
Zhang1, Jie-Sheng Huang1, Chi-Ming Che1
1The University of Hong Kong
Bioconjugation of proteins has emerged as a useful tool in the study of biological
systems. There is an increasing need to develop new synthetic technologies for the
bioconjugation reaction of proteins, and metal-catalyzed site-selective modification
of proteins has attracted considerable interest in recent years. We have developed
a ruthenium glycosylated porphyrin-catalyzed carbenoid transfer reaction for the site-selective
modification of proteins. We firstly applied the catalysis to the selective modification
of the N-terminus of peptides. By using ruthenium glycosylated porphyrin as catalyst,
the N-terminus of a number of peptides can be modified through carbenoid N-H bond
insertion in aqueous media with moderate to excellent conversion. The reaction is
highly selective, for example, the reaction with YTSSSKNVVR, which contains various
types of oxygen–hydrogen and nitrogen–hydrogen bonds possibly available for carbenoid
insertion, catalyzed by the ruthenium glycosylated porphyrin gave the N-terminal-modified
product with >99% conversion and without the formation of other modified peptides
including doubly modified and oxygen–hydrogen bond insertion products. We next extended
the N-terminal modification method to proteins. Eventually success was attained in
the modification of RNase A and insulin. The reaction of RNase A with a diazoacetate
mediated by ruthenium glycosylated porphyrin gave corresponding N-terminal-modified
protein with 65% conversion. We also achieved a bioconjugation to ubiquitin via ruthenium
glycosylated porphyrin-catalyzed alkene cyclopropanation in aqueous solution in two
steps: (1) incorporation of an alkenic group by the reaction of N-hydroxysuccinimide
ester with ubiquitin and (2) cyclopropanation of the alkene-tethered Lys6 ubiquitin
with the fluorescent labeled diazoacetate in the presence of a catalytic amount of
ruthenium glycosylated porphyrin. The corresponding cyclopropanation product was obtained
with ∼ 55% conversion based on MALDI-TOF mass spectrometry. In conclusion, we developed
a ruthenium porphyrin-catalyzed site-selective modification of peptides and proteins
in aqueous media. The method provides an entry to new bioconjugation reactions for
protein modifications using metalloporphyrins as catalysts.
PB-043
Modulating the affinities of phophopeptides to human Pin1 WW domain using 4-substituted
proline derivatives
Jia-Cherng Horng1, Kuei-Yen Huang1
1Department of Chemistry, National Tsing Hua University
Human Pin1 is involved in cancer developments and has been a pharmaceutical target.
Thus, finding a high affinity inhibitor of Pin1 has become an attractive topic. The
WW domain of human Pin1 can recognize the phosphoserine/phosphothreonine-proline (pS/pT-P)
motifs, while its PPIase domain catalyzes the cis/trans isomerization of prolyl bonds
to regulate the cell cycle. Here we incorporated a series of 4-substituted proline
derivatives into the phosphopeptides and investigated their affinities to the WW domain
of Pin1 to develop better inhibitors of Pin1. Based on the ligand Myt1-T412 [PPA(pT)PP],
we synthesized several phosphopeptides in which proline residue in the pT-P motif
was replaced with various 4-substituted proline derivatives. Isothermal titration
calorimetry and fluorescence anisotropy analyses show that the replacement of proline
by (2S,4R)−4-fluoroproline increases the binding affinity of the peptide. Circular
dichroism measurements suggest that a more PPII-like structure of phosphopeptides
make them bind to the WW domain more tightly. Chemical shift perturbation experiments
also indicate that (2S,4R)−4-fluoroproline interacts with Trp34 of the WW domain in
the binding site. Results of molecular modeling further suggested that a strong C-H…
π interaction induced by (2S,4R)−4-fluoroproline is important in enhancing the affinity
of the peptide to the WW domain. The results of our present study provide new valuable
information for designing and developing effective inhibitors of human Pin1.
PB-044
Applying an analytical ultracentrifuge equipped with fluorescence detection to the
study of polyglutamine aggregation in Caenorhabditis elegans
Bashkim Kokona1, Carrie A. May2, Nicole R. Cunningham1, Franklin J. Garcia1, Kathleen
M. Ulrich1, Christine M. Roberts4, Christopher D. Link4, Walter F. Stafford3, Thomas
M. Laue2, Robert Fairman1
1Department of Biology, Haverford College, 2Department Of Molec., Cell., and Biomed.
Sci., University of New Hampshire, 3Boston Biomedical Research Institute, 4Integrative
Physiology, University of Colorado Boulder
This work aims to explore the heterogeneity of aggregation of polyglutamine fusion
constructs in crude extracts of an animal model system, using transgenic Caenorhabditis
elegans animals. The work takes advantage of the recent technical advances in fluorescence
detection as coupled with analytical ultracentrifugation. Further, new methods of
analysis of sedimentation velocity experiments, such as gravitational sweep experiments,
are applied to improve the resolution of the measures of heterogeneity over a wide
range of sizes. The focus here is to test the ability to measure sedimentation of
polyglutamine aggregates in complex mixtures, as a prelude to future studies that
will explore the effects of genetics and environment on aggregation and toxicity.
Using sedimentation velocity methods, we can detect a wide range of aggregates, ranging
from robust analysis of the monomer species, through an intermediate and quite heterogeneous
population of oligomeric species, and all the way up to detecting species that likely
represent intact inclusion bodies based on comparison to an analysis of fluorescent
punctates in living worms by confocal microscopy. Our results support the hypothesis
that misfolding of expanded polyglutamine tracts into insoluble aggregates involves
transition through a number of stable intermediate structures, a model that accounts
for how aggregation can be at the same time toxic and protective. An understanding
of the details of small, intermediate, and large-scale aggregation for polyglutamine
sequences, as found in neurodegenerative diseases such as Huntington’s Disease, will
help to more precisely identify which aggregated species may be involved in toxicity
and disease.
PB-045
Probing the selectivity of peptide carrier protein-tailoring enzyme interactions using
analytical ultracentrifugation
Robert Fairman1, Bashkim Kokona1, Emily S. Winesett2, Alfred N. von Krusenstiern2,
Max J. Cryle3, Louise K. Charkoudian2
1Department of Biology, Haverford College, 2Department of Chemistry, Haverford College,
3Max Planck Institute for Medical Research
Bacteria and fungi use non-ribosomal peptide synthetases (NRPSs) to produce peptides
of broad structural diversity and biological activity. The impressive diversity of
non-ribosomal peptides originates in part from the action of tailoring enzymes that
modify single amino acids and/or the mature peptide. Studying the interplay between
tailoring enzymes and the peptide carrier proteins (PCPs) that anchor and present
the substrate is challenging because the transient nature of the protein-protein interactions.
Using sedimentation velocity (SV) methods, we studied the collaboration between PCP
and cytochrome P450 enzyme that is crucial for the installation of β-hydroxylated
amino acid precursors in the biosynthesis of the depsipeptide skyllamycin. We show
that sedimentation velocity (SV) is an ideally suited method for a quantitative exploration
of PCP-enzyme equilibrium interactions. Our results suggest that the PCP itself and
the presence of substrate covalently tethered to the PCP together facilitate productive
PCP-P450 interactions, thereby revealing one of nature’s intricate strategies for
installing interesting functionalities using natural product synthases.
PB-046
Uridine Monophosphate Synthase: Architecture Versatility in the Service of Late Blight
Control
Francisco Tenjo Castaño1,2, Manuel Garavito1,2, Leonor García1,2, Silvia Restrepo2,
Barbara Zimmermann1
1Biochemistry and Molecular Biology Research Group, Universidad de los Andes., 2Mycology
and Plan Pathology Laboratory, Universidad de los Andes
Uridine monophosphate synthase (UMPase), a bifunctional enzyme in the de novo pyrimidine
biosynthetic pathway, is a protein comprised of orotate phosphoribosyl transferase
(OPRTase) and orotidine monophosphate decarboxylase (ODCase). Different fusion orders
of the two domains have been documented to exist in nature. In some organisms OPRTase
and ODCase are monofunctional proteins, and act as a complex. Here, UMPase from Solanum
tuberosum (potato) and from Phytophthora infestans (an oomycete) were examined. P.
infestans causes late blight disease in S. tuberosum, destroying crops and increasing
production costs. Since pyrimidines are fundamental cellular components, we have proposed
that UMPase could serve as a target to control P. infestans infection. The enzymes
from P. infestans and S. tuberosum differ in their fusion order of OPRT and ODC. The
study of these two UMPase could facilitate the design of species-specific inhibitors,
and might shed light on the effect of fusing UMPase domains in one order or the other.
To this end we carried out bioinformatic and biochemical characterization of the enzymes.
Sequence analyses showed 20 residue differences among the P. infestans UMPase sequences
from three strains: 4084, 1306 and T30-4. Strain T30-4 was found to have a duplicated
UMPase, but neither sequence corresponded to the ones predicted previously from the
genome. A recombinant UMPase from 4084 strain was expressed in bacteria and purified
but it showed low solubility and was inactive in vitro. The recombinant UMPase from
the 1306 strain complemented both OPRTase and ODCase deficient E. coli strains. A
soluble, active, recombinant protein was expressed and purified in the presence of
high salt and the product UMP (specific activity ≈ 0.2 μmol min-1 mg-1). The sequence
SKQ was found at the C-terminus of the P. infestans UMPase sequences and resembles
a peroxisome signal peptide (SKL). The predicted hydrophobicity of this UMPase and
its architecture (OPRT at the C-terminus and ODC at the N-terminus) resembles that
of the UMPase from Leishmania donovani, which has been localized to the peroxisome.
We suggest that P. infestans UMPS could also be located in this organelle. In contrast
to the oomycete enzyme, S. tuberosum UMPase is highly soluble, and has a higher specific
activity (Vmax= 8.8 μmol min-1 mg-1). We measured the kinetic parameters KM(orotate)=
16.2 μM, KM(PRPP)= 25.5 μM, and found that it exhibited product inhibition by pyrophosphate.
In conclusion, the different architectures of the two UMPS might be related to distinct
biochemical characteristics, further supporting this protein as a good candidate for
P. infestans control.
PB-047
Three Antimicrobial Peptides: MD Simulation Studies Supporting Experiment
Walter Scott1, Vivien Schubert2, Andi Mainz2, Suzana K. Straus1, Roderich Suessmuth2
1Department of Chemistry, University of British Columbia, 2Institut fuer Chemie, Technische
Universitaet Berlin
We present computer simulation studies of three different antimicrobial peptides we
have been studying by MD computer simulation in collaboration with experimentalists.
The first is Daptomycin, a potent lipopeptide currently licensed to treat infections
caused by multi-drug-resistent bacteria. The mechanism of action of Daptomycin is
currently not completely understood. We have solved the NMR structure of this molecule,
and attempted to determine the size of its oligomer by small angle neutron scattering
(SANS) supported by computer simulation. Feglymycin is a 13-amino-acid peptide with
a high percentage of unusual amino acids such as 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine.
Feglymicin inhibits MurA and MurC enzymes which are involved in bacterial peptidoglycan
synthesis, while also displaying anti-HIV activity by interaction with the viral envelope
protein gp120. A previous X-ray structure shows the molecule forming a dimer. Here,
the molecule was studied by NMR in water and DMSO. In water, the molecule is clearly
at least a dimer, while in DMSO it is a monomer. We have performed NOE refinement
simulations in order to elucidate a structure, however, due to a lack of long-range
NOE contacts, a unique structure cannot be determined. Labyrinthopeptin A2 is a lantibiotic
that contains labionin, a unique carbacyclic posttranslationally modified amino acid
that links the protein backbone in three different locations. Labyrinthopeptin A2
has shown promising activity as a pain killer. Starting from the X-ray structure,
we present results from the first MD simulation studies of this unique peptide. Because
of the extensive cross-linking, this peptide is observed to be highly rigid in its
native form. Simulation results of mutants are also presented.
PB-048
Studying the Outer Membrane ß-barrel Protein LptD, the Target of a New Peptidomimetic
Antibiotic
Katja Zerbe1, Gloria Andolina1, Laszlo Bencze1, Kerstin Moehle1, John A. Robinson1
1Department of Chemistry, University Zurich
Antibiotics with new mechanism of action are urgently required to combat the growing
health threat posed by resistant pathogenic microorganisms. Here we report the discovery
of a new peptidomimetic antibiotic (L27-11), which is active with a minimum inhibitory
concentration (MIC) in the low nanomolar range, only against Pseudomonas sp., and
with a non-membrane-lytic mechanism of action. A drug target identified both in a
forward genetic screen for resistance determinants and by photoaffinity labeling is
the ß-barrel protein LptD, which plays an important role in LPS transport and the
outer membrane biogenesis. The X-ray structure of LptD in complex with LptE from Shigella
flexneri shows a 26 stranded β-barrel linked to a periplasmatic N-terminal jelly-roll
domain. Interestingly the homology model structure for LptD from Pseudomonas shows
a significant difference: an insertion of around 100 amino acids in the N-terminal
domain. The results of our attempts to purify and characterize this large outer membrane
protein and to determine the binding site of the peptidomimetic antibiotic will be
shown.
Fig. 1
PB-049
Structure and catalytic properties of peptides based on sequences of P-loop from ATP
binding domains
Wioletta Zmudzinska1, Marcel Thiel1, Stanislaw Oldziej1
1IFB, University of Gdansk and Medical University of Gdansk
The theory of how life on Earth begun still remains unclear. Nevertheless, according
to some theories, at the beginning level proteins did not emerge as a complex globular
forms as know today. At the times, when solely RNA molecules stored both genetic information
and catalyzed the chemical reactions in primitive cells, peptides acted as a proteins
nowadays [1,2]. Literature postulate that the possible role of primordial short peptides
was to catalyze reactions in RNA-world, as they possess an excellent ability to self-assemble
into well-ordered nanostructures [3,4]. Elementary Functional Loops (EFLs) can be
considered as a small structures (blocks) having specific signatures and providing
functional residues important for binding/activation as well as principal chemical
transformation steps of the enzymatic reaction [5]. P-Loop EFL is a widespread structure
across vast majority of protein families such as motor domains, AAA+, RecA, PEPCK
and many others. Sequential alignment of these protein families reveals existence
of a conserved P-loop motif, that is able to bind ATP molecule. We investigated the
structure and ATPase activity of peptides, which sequences possessed strongly conserved
GXGK[T/S] motif from P-loop. The goal of our work was to check if peptides corresponding
to the most conserved P-loop motif fragment are able to bind and hydrolyze ATP molecule.
All peptides under study were chemically synthesized and their structures was investigated
by NMR spectroscopy. The ability to bind ATP molecules was analyzed by using HPLC
chromatography. Results of our study show, that peptides with conserved P-Loop motif
have a suitable structures to promote binding of the molecules with phosphate group,
but cannot accelerate pyrophosphate hydrolysis process. Conference participation for
W. Ż. supported by the FP7 project MOBI4Health (grant agreement no 316094). Computational
resources were provided by the Informatics Center of the Metropolitan Academic Network
(IC MAN TASK) in Gdansk, Poland.
[1] Carny O & Gazit E (2005) A model for the role of short self-assembled peptides
in the very early stages of the origin of life. Faseb J 19(9):1051-1055
[2] Carny O & Gazit E (2010) Creating prebiotic sanctuary: self-assembling supramolecular
Peptide structures bind and stabilize RNA. Orig Life Evol Biosph 41(2):121-132
[3] Gazit E (2007) Self-assembled peptide nanostructures: the design of molecular
building blocks and their technological utilization. Chem Soc Rev 36(8):1263-1269
[4] Kol N, et al. (2005) Self-assembled peptide nanotubes are uniquely rigid bioinspired
supramolecular structures. Nano Lett 5(7):1343-1346
[5] Goncearenco A & Berezovsky IN (2010) Prototypes of elementary functional loops
unravel evolutionary connections between protein functions. Bioinformatics 26(18):i497-503
PB-050
The atp-binding site of CK2 carries two regions with antagonistic electrostatic potential
that atracts charged ligands
Maria Winiewska1, Jarosław Poznański1
1Institute of Biochemistry and Biophysics Polish Academy of Sciences
CK2 is a ubiquitous serine/threonine protein kinase, being one of the most pleiotropic
of all protein kinases1. CK2 plays a key role in cell growth, differentiation, cell
death and survival, and become the therapeutic target in cancer treatment, since its
level is significantly increased in cancer cells2. Halogenated ligands have been widely
developed as potent inhibitors of protein kinases. Among them 4,5,6,7-tetrabromobenzoteriazole
(TBBt) is one of the first potent and selective inhibitor of CK2α, directed towards
the conserved ATP binding site3. To assess contribution of electrostatic interactions
to the specificity and strength of binding of multi halogenated inhibitors by a protein
kinase, we have studied interaction between CK2α and nine benzotriazole derivatives,
representing all possible patterns of halogenation on the benzene ring. Herein, we
present results that support existence of two alternative regions that are involved
in ligand binding. Aspartic acid 175 is known for its function in coordination of
a Mg2+ ion, which is required for ATP binding 4. Asp175 has been identified in crystal
structure of CK2:TBBt complex (PDB1j91, Fig. 1) as the charged residue closest to
TBBt. There is also Lys68 proximal to TBBt, interaction with which may favor anionic
form of ligands5 (pK for TBBt <5), however it is involved in the intramolecular salt
bridge, and thus its mutation may significantly change stability of the protein.
Crystal structure of TBBt complexed with CK2 (PDB:1j91). Residues with a distance
to TBBt (Magenta) shorter than 5A are shown. Red residue is negatively charged, blue
ones are protonated.
Fig. 1
Comparison of Kdiss values determined for ligands at pH 8 and at pH 7 shows that strength
of the complex significantly varies upon deprotonation of the triazole ring. This
confirms former hypothesis that a negatively charged ligands cluster at the ATP binding
site region proximal to Lys685, which is beneficial both to the specificity and to
strength of the binding. We have also observed for the tested ligands variations in
their binding to either wild type protein and its D175N mutant (with less negative
charge distributed over ATP binding site). All ligands displaying higher pKa for dissociation
of the triazole proton bind to the mutant visibly weaker than to the wild-type protein.
Altogether reveals the predominance electrostatic intermolecular interactions. Although,
negatively charged ligands most probably cluster at the ATP-binding site proximal
to Lys68, beneficial for the strength of binding, the less dissociated forms are favored
due to unfavorable interactions of the anionic form of ligands with Asp175.
Acknowledgments:
This work was supported by the Polish National Science Centre grant 2012/07/B/ST4/01334.
1. L. A. Pinna, Protein Kinase CK2, John Wiley & Sons, New York, NY, 2012.
2. Tawfic, S. et.al, Histology and Histopathology, 2001, 16, 573-582.
3. G. Cozza, L. A. Pinna and S. Moro, Current Medicinal Chemistry, 2013, 20, 671-693.
4. Battistutta R et al., Chem Biol, 2005, 12, 1211-1219.
5. Battistutta R et al., ChemBiochem, 2007, 8, 1804-1809.
PB-052
NMR Solution Structure Elucidation of Phenol Soluble Modulins; Virulence Factors in
Staphylococcus aureus
Kaitlyn M. Towle1, Christopher T. Lohans2, Marco J. van Belkum1, Mark Miskolzie1,
John C. Vederas1
1University of Alberta, 2University of Oxford
Phenol soluble modulins (PSMs) are a class of toxins isolated from the Staphylococcus
sp.1 Of particular interest are the PSMs, produced by the infectious Methicillin Resistant
Staphylococcus aureus (MRSA). MRSA infections have been on the rise through both community
associated MRSA (CA-MRSA) and hospital associated MRSA (HA-MRSA) strains. 1 There
are many virulence factors produced by these strains, many of which are encoded on
mobile genetic elements. 2 PSMs are of specific interest because these virulence factors
are encoded on the core genome of the bacteria and therefore all strains of staphylococci
bacteria produce some variation of PSMs with a variety of biological functions.2 The
specific mechanism by which PSMs act as virulence factors has been poorly understood
until recently. Biological functions of PSMs include cell lysis, biofilm formation
and the ability to kill neutrophils after phagocystosis.1 These toxins are of special
interest to our research group due to their genetic similarities to certain bacteriocins,
namely leaderless bacteriocins. 3 Both groups of peptides are ribosomally synthesized
with a N-terminal formyl methionine and secreted from the bacteria by ATP-binding
cassette (ABC) transporters without any leader sequence or signal peptide. ABC transporters
may also play a role in immunity towards PSMs and leaderless bacteriocins. These similarities
led our group to investigate the solution structure of these peptides through nuclear
magnetic resonance (NMR). Isolating PSMs from the producer organisim, S. aureus, typically
involves lengthy extractions and low yields. 4 For these reasons, we opted to chemically
synthesize the desired peptides using solid phase peptide synthesis (SPPS). Utilizing
a variety of SPPS techniques, PSM α1 and PSM α3 were successfully synthesized, however,
due to the hydrophobic nature of PSM β2, an alternate genetic approach was devised
to isolate PSM β2. Formation of a fusion protein between PSM β2 and the small ubiquitin
like modifier (SUMO) protein allowed for heterologous expression. Upon cleavage of
the fusion protein with SUMO protease, and subsequent purification and isolation of
the cut peptide, PSM β2 was obtained. As previously reported, the PSMs were found
to be alpha-helical in structure inducing solvents. 5 A series of 2 dimensional (2D)
NMR experiments were ran to determine chemical shift assignments and to obtain NOE
data. Importing the chemical shift assignments and NOE data into the structure calculating
software, CYANA, we were able to elucidate the solution structure of PSM α1 and PSM
α3 and we are currently working towards the elucidation of PSM β2. The synthesis,
isolation, characterization and solution structures of the aforementioned PSMs will
be discussed here. 1. Peschel, A.; Otto, M. Nature Reviews Microbiology, 2013, 11,
667. 2. Cheung, G.Y.C.; et al. FEMS Microbiol Rev. 2014, 38, 698. 3. Cintas, L. M.;
et al. J. Bacteriol. 1988, 180. 4. Wang, R.; et al. Nature Medicine 2007, 13, 1510.
5. Laabei, M.; et al. Biochimica et Biophysica Acta 2014, 1838, 3153.
PB-053
Mitochondrial iron as a potential therapeutic target in friedreich’s ataxia neurodegeneration:
desferioxamine-peptide conjugate
Roxana Yesenia Pastrana Alta1, Maria Teresa Machini2, Breno Pannia Espósito1
1Laboratory for Bioinorganic Chemistry and Metallodrugs, Instituto de Química, Un,
2Laboratory of Peptide Chemistry, Instituto de Química, Universidade de São Paulo
Transition metals are critical for enzyme function and protein folding, but their
excess can mediate neurotoxic oxidative processes [1]. As, energy production involves
oxidative phosphorylation, a process requiring a continuous flow of electrons, mitochondria
are particularly vulnerable to oxidative damage [2]. As such, mitochondria are the
major sites of Reactive Oxygen Species (ROS) generation, which are produced as byproducts
of the electron transport chain. Since free iron and certain ROS can engage into potentially
deleterious processes such as Fenton reaction, mitochondrial iron homeostasis must
be tightly controlled, and dysregulation of iron metabolism in this organelle has
been associated with various diseases, including Friedich´s ataxia (FA), Alzheimer’s,
and other neurodegenerative disorders [3]. Engineering an efficient mitochondria-targeting,
cell-permeable vector is a challenge due to the fact that mitochondrion is impermeable
to a wide range of molecules. The development of delivery vectors has been made possible
by a greater understanding of mitochondrial structure and chemical features of molecules
that selectively localize within this organelle. From these findings, two generalized
requirements for mitochondrial localization are delocalized positive charge and lipophilicity
[4, 5]. Targeting iron in this organelle is proposed as a means to ameliorate FA symptoms.
Desferrioxamine (DFO) is a bacterial siderophore with high affinity for iron, but
low cell penetration. We prepared conjugates of DFO with Mitochondria Penetrating
Peptides and studied their iron-binding characteristics in vitro. The lipophilic and
charged peptides TAT49-57 (H-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH)[6], 1A (H-Cha-Arg-Cha-Lys-Cha-Arg-Cha-Lys-NH2)[6],
SS-02 (H-Dmt-Arg-Phe-Lys-NH2)[7] and SS-20 (H-Phe-Arg-Phe-Lys-NH2) [7], are known
to permeate cytosolic and mitochondrial membranes. They were prepared and conjugated
to DFO in solid-phase [8], an alternative synthetic route. Once detached from the
resin, fully deprotected, purified and characterized by means of LC/MS and aminoacid
analysis, it was observed that the DFO-conjugated peptides displayed iron-binding
abilities identical to the free chelator DFO. DFO-conjugated peptides were also able
to quench the iron-catalysed oxidation of ascorbate (a model of oxidative stress in
plasma of iron-overloaded patients), as probed by a high throughput fluorimetric method
[9,10]. These results indicate that our synthesis and conjugation strategy were successful
in preserving the iron-binding moiety and the antioxidant ability of the free chelator
DFO.
PB-054
The proteolytic activity and oligomerization status of the human HtrA3 protease functioning
as a tumor suppressor
Przemyslaw Glaza1, Tomasz Wenta1, Jerzy Osipiuk3, Agnieszka Kowalska1, Ewa Gebal1,
Dorota Zurawa-Janicka1, Adam Lesner4, Barbara Lipinska1
1Department of Biochemistry, Faculty of Biology, University of Gdansk, 2Midwest Center
for Structural Genomics, Argonne National Laboratory, 3Structural Biology Center,
Biosciences Division, Argonne National Laboratory, 4Department of Biochemistry, Faculty
of Chemistry, University of Gdansk
HtrA3 protease belongs to the high-temperature requirement A (HtrA) family of serine
proteases which take part in cellular stress response including heat shock, inflammation
and cancer. HtrA3 is composed of an N-terminal domain not required for proteolytic
activity, a central serine protease domain and a C-terminal PDZ domain. The latter
serves as a substrate or regulator binding domain and may participate in oligomerization.
HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a
stretch of 7 C-terminal amino acid residues, unique for this isoform. Down-regulation
of HtrA3 in tumors, shown by other groups and us, suggests HtrA3s involvement in oncogenesis
[1]. HtrA3 acts as a proapoptotic protein and is suggested to function as a tumor
suppressor. It promotes cytotoxicity of etoposide and cisplatin in lung cancer cell
lines [2,3]. To date, HtrA3 has been poorly characterized from the biochemical point
of view, mainly due to the fact that it is difficult to purify recombinant HtrA3.
We were able to express in bacterial system and purify HtrA3 in quantities sufficient
to perform structural studies. The aim of this study was to characterize and compare
the proteolytic properties and quaternary structure of the HtrA3 isoforms. Both studied
isoforms lacked the N-terminal domain. HtrA3 with the PDZ domain removed (HtrA3-ΔPDZ)
and HtrA3S (HtrA3S) were fully active at a wide range of temperatures and their substrate
affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3
activity. As determined by size exclusion chromatography, HtrA3 formed stable trimers
while both HtrA3-ΔPDZ and HtrA3S were monomeric. This suggests that the presence of
the PDZ domain, unlike in other human HtrAs (HtrA1 and HtrA2), influences HtrA3 trimer
formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect
on activity and oligomerization [4].
1. Skórko-Glonek J et al. (2013) Curr Pharm Des 19: 977-1009.
2. Beleford D et al. (2010) Clin Cancer Res 16: 398-409.
3. Beleford D et al. (2010) J Biol Chem 285:12011-27.
4. Glaza P et al. (2015) PLoS ONE doi: 10.1371/journal.pone.0131142.
PB-055
Cyclodextrins moderately affects binding of halogenated benzotriazoles by protein
kinase CK2
Katarzyna Kuciñska1, Maria Winiewska1 Jarosław Poznański1
1Institute of Biochemistry and Biophysics Polish Academy of Sciences
Cyclodextrins (CDs) are cyclic oligosaccharides that have been recognized as useful
pharmaceutical excipients. In aqueous solution CDs are capable to form complexes with
various ligands, hosting inside their cavity either a whole molecule, or part of a
ligand. Inclusion complexes with CDs offers a variety of physicochemical advantages
over the biologically active ligands, including the improved aqueous solubility, solution
stability or an increase of bioavailability. CK2 is an ubiquitous, highly pleiotropic
and constitutively active Ser/Thr protein kinase. Halogenated benzotriazoles have
been developed as potent and selective inhibitors of this enzyme. The interaction
of the catalytic domain of human protein kinase CK2 with a series of brominated ligands,
which represent all possible patterns of halogen substitutions to the benzene ring
of benzotriazole, was previously studied by microscale thermophoresis (MST) [1]. This
method alloweddetermination of binding affinities for seven ligands, all of which
were found consistent with the values determined independently by isothermal titration
calorimetry (ITC). However, a very limited aqueous solubility of some brominated benzotriazoles
may decrease their bioavability, thus affectingtheir apparent activity[2]. To overcome
this limitation, the aqueous solubility of halogenated benzotriazoles in the presence
of cyclodextrins has been tested. The formation of inclusion complexes with β-cyclodextrin
(β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD) and γ-cyclodextrin (γ-CD) in aqueous
solutions, followed by UV–Vis spectroscopy, substantially improved the solubility
of TBBt and its derivatives. The interaction between protein kinase CK2 and cyclodextrins,
and also with their inclusion complexes with halogenated benzotriazoles, was followed
with the aid of the microscale thermophoresis. The results obtained clearly demonstrate
that the binding of halogenated benzotriazoles by CK2 is only moderately affected
by cyclodextrins.
[1] M. Winiewska, K. Kucińska, M. Makowska, J. Poznański, D. Shugar, Biochim Biophys
Acta. 2015, doi: doi: 10.1016/j.bbapap.2015.04.004
[2] R. Wąsik, P. Wińska, J. Poznański, D. Shugar, J. Phys.Chem. B, 116, (2012), 7259-7268;
Acknowledgments:
This work was supported by the Polish National Centre for Science grant 2012/07/B/ST4/01334.
The equipment used was sponsored in part by the Centre for Preclinical Research and
Technology (CePT), a project co-sponsored by European Regional Development Fund and
Innovative Economy, The National Cohesion Strategy of Poland.
PB-056
Antibody Activation using DNA-Based Logic Gates
Maarten Merkx1, Brian Janssen1, Martijn van Rosmalen1, Lotte van Beek1
1Laboratory of Chemical Bology, Eindhoven University of Technology
Oligonucleotide-based molecular circuits offer the exciting possibility to introduce
autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics.
Here we introduce bivalent peptide–DNA conjugates as generic, noncovalent, and easily
applicable molecular locks that allow the control of antibody activity using toeholdmediated
strand displacement reactions. Employing yeast as a cellular model system, reversible
control of antibody targeting is demonstrated with low nm concentrations of peptide–DNA
locks and oligonucleotide displacer strands. Introduction of two different toehold
strands on the peptide–DNA lock allowed signal integration of two different inputs,
yielding logic OR- and AND-gates. The range of molecular inputs could be further extended
to protein-based triggers by using proteinbinding aptamers.
PB-057
Insights of a novel kind of cell wall binding domain that cleaves the peptidoglycan
muropeptide: the CW_7 motif
Noemí Bustamante1,3, Manuel Iglesias, Noella Silva-Martín, Isabel Uson, Pedro García,
Juan Hermoso, Marta Bruix, Margarita Menéndez
1Institute of Physical-Chemistry ‘Rocasolano’, CSIC, 2Institute of Physical-Chemistry
‘Rocasolano’, CSIC, 3Ciber of Respiratory Diseases (CIBERES), 4Center of Biological
Research (CIB), CSIC, 5Institucio Catalana de Recerca i Estudis Avançats, CSIC-IBMB
Enzybiotics constitute a hopeful alternative to current treatments to fight against
bacterial infections. Phage endolysins are consider as enzybiotics due to their capacity
to cleave the peptidoglycan (PG) of Gram-positive bacteria in a generally species-specific
manner and kill bacteria when exogenously added (1,2). The Cpl-7 endolysin, a lysozyme
encoded by the pneumococcal Cp-7 bacteriophage, is a remarkable exception among all
the PG hydrolases produced by Streptococcus pneumoniae and its bacteriophages due
to its capacity of degrading pneumococcal cell walls containing either choline or
ethanolamine (3, 4). This fact confers to Cpl-7 the advantage of displaying a broader
microbicide spectrum comparing to choline binding proteins (5). This behavior results
from the acquisition of a cell wall binding module (CWBM) made of three identical
repeats of 48 amino acids each (CW_7 motifs), with unknown specificity and totally
unrelated with the choline-binding motives present in pneumococcal hydrolases. Interestingly,
CW_7 repeats have been identified in many putative proteins potentially involved in
cell wall metabolism (Pfam entry: PF08230) from different species of Gram positive
and Gram negative bacteria, and some bacteriophages (6). Preliminary studies of thermal
stability in presence of a small cell wall structural-analogue (GlcNAc-MurNAc-L-Ala-D-IsoGln)
point to the muropeptide as the cell wall target recognized by CW_7 motifs (7). In
this communication we have gone in depth in the characterization of CW_7 repeats.
We present the first crystal structure of the CW_7 motif, which reveals a three-helical
bundle folding. Using STD_NMR spectroscopy the epitope of binding of the disacharide
dipeptide to this repeats has been identified. Interestingly, the β anomer of the
MurNAc moiety, the form present in the peptidoglycan, seems to be preferentially recognized
with respect to the α anomer. Finally, a docking model of the complex CW_7/GMDP compatible
with STD results was built allowing to identify the major contacts between the protein
and the muropeptide and to propose the relevant role of a conserved Arginine residue
in this interaction.
1. FISCHETTI, VA. Bacteriophage lytic enzymes: novel anti-infectives, Trends Microbiol.,
13, 491-496.
2. HERMOSO, J.A., et al. Taking aim on bacterial pathogens: from phage therapy to
enzybiotics. 2007. Curr. Opin. Microbiol. 10. 461-472.
3. LÓPEZ, R. and GARCÍA E. Recent trends on themolecular biology of pneumococcal capsules,
lytic enzymes, and bacteriophage. 2004. FEMS Microbiol. Rev. 28, 553-580.
4. GARCÍA, P. et al. Modular organization of the lytic enzymes of Streptococcus pneumoniae
and its bacteriophages. 1990. Gene, 867, 81-88.
5. DÍEZ-MARTINEZ, R. et al. Improving the lethal effect of Cpl-7, a pneumococcal phage
lysozyme with broad bactericidal activity by inverting the net charge of its cell
wall-binding module.2013. Antimicrob. Agents Chemother. 57(11): 5355
6. BUSTAMANTE, N. et al. Cpl-7 a lysozyme encoded by a pneumococcal bacteriophage
with a novel cell wall-binding motif. 2010. J. Biol. Chem. 285,33184-33196.
7. BUSTAMANTE, N. et al. The Cpl-7 endolysin from Cp-7 pneumococcal bacteriophage:
thermal stability and cell wall-targeting specifity. 2012. PLOS ONE, 7 (10):e46654
PB-058
Engagement of the ClpS NTE by the ClpAP machinery inhibits substrate recognition and
processing
Amaris Torres-Delgado1, Robert T. Sauer1, Tania A. Baker1,2
1Department of Biology, Massachusetts Institute of Technology, 2Howard Hughes Medical
Institute
Energy-dependent AAA+ proteases carry out regulated proteolysis to ensure protein
quality control and post-translational regulation of many cellular processes. Control
of proteolysis occurs primarily at the level of substrate recognition, which can be
modulated by adaptor proteins. The ClpS adaptor protein enhances and inhibits degradation
of different classes of substrates, and thus triggers a specificity switch in ClpA.
Whereas the mechanism for substrate delivery by ClpS has been described in detail,
the inhibition mechanism is poorly understood. We show that ClpS inhibits ssrA substrate
recognition and processing, instead of simply preventing substrate binding. We demonstrate
that ClpA engagement of the ClpS N-terminal extension (NTE) is necessary, and may
even be sufficient, for inhibition. In addition, we find that inhibition of substrate
processing requires a longer NTE, as compared to inhibition of substrate recognition.
Interestingly, the NTE length required for inhibiting substrate processing is also
necessary for suppression of the ClpA ATPase rate. Furthermore, preliminary data suggests
that ClpS slows down substrate translocation. These results support a model where
there is an ssrA•ClpA•ClpS inhibitory complex in which the ClpA pore engages the ClpS
NTE. This engagement of the NTE causes suppression of ATPase activity, and therefore
slower substrate translocation and processing. This model illustrates how an adaptor
protein can inhibit recognition of one type of substrate while efficiently promoting
degradation of a different substrate.
PB-059
Single-molecule assay development for studying Human RNA Polymerase II Promoter-Proximal
Pausing
Yazan Alhadid1, Benjamin Allen2, Sangyoon Chung1, Dylan Taatjes2, Shimon Weiss1
1University of California, Los Angeles, 2Univeristy of Colorado Boulder
Single-molecule assay development for studying Human RNA Polymerase II Promoter-Proximal
Pausing Yazan Alhadid, Benjamin Allen, Sangyoon Chung, Dylan Taatjes, Shimon Weiss
Abstract: Promoter-proximal RNA Polymerase II (PolII) pausing has been shown to play
a significant role in transcription regulation of elongating PolII complexes in a
large number of metazoan and mammalian genes(1). The traditional understanding of
transcription regulation in mammals involved controlling PolII recruitment to promoters
and controlling initial steps at the promoter, including pre-initiation complex formation
and promoter escape. Most works investigating promoter-proximal PolII pausing have
employed chromatin immunoprecipitation followed by sequencing to determine PolII localization
or in vitro transcriptional assays using nuclear extracts analyzed with radio-active
gel electrophoresis. In order to gain greater mechanistic insight into the regulation
of promoter-proximal PolII pausing, we have been developing a diffusion-based single-molecule
method using alternating laser excitation on the micro-second timescale (µsALEX).
The method detects RNA transcripts generated by a reconstituted human PolII system
in vitro using complementary doubly dye-labeled single-stranded DNA (ssDNA) probes.
The human gene HSPA1B for heat shock protein 70 (Hsp70) is used as a model system
due to its extensive characterization in drosophila. The method would provide a rapid,
sensitive and robust avenue to screen for protein factors regulating promoter-proximal
PolII pausing. Controlling of the PIC composition using the reconstituted system allows
for dissection of the functional roles of different PIC components in facilitating
regulation of PolII pausing. We have demonstrated the hybridization of double dye-labeled
ssDNA probe to complementary ssDNA mimicking RNA transcripts and to transcripts generated
with bacterial RNA polymerase. Also, a functional reconstituted human PolII system
has been verified using radioactive polyacrylamide gel electrophoresis of transcripts
from in vitro transcription assays.
1. H. Kwak, J. T. Lis, Control of transcriptional elongation. Annu. Rev. Genet. 47,
483–508 (2013).
PB-060
Structural characterization of Plasmodium falciparum CCT and fragment-based drug design
approach for targeting phospholipid biosynthesis pathway
Ewelina Guca1, François Hoh2, Jean-François Guichou2, Henri Vial1, Rachel Cerdan1,
1DIMNP, UMR 5235, University of Montpellier, 2Centre de Biochimie Structurale, INSERM
Malaria is a major global health problem. In 2013, there were an estimated 128 million
case of malaria and 584 000 deaths, most of them children under 5 years old [1]. Among
the 5 malaria species that affect humans, Plasmodium falciparum is the most deadly
form. Since no efficient vaccine is available yet, the fight against malaria includes
vector control, protection from mosquito bites and artemisinin combined therapy. However,
resistances to all known treatments have been observed. Therefore, new antimalarial
strategies involving novel targets and new mechanisms of action are needed. During
its life cycle, in erythrocytic stage, which causes all the malaria symptoms, Plasmodium
falciparum relies on phospholipids to build the membranes necessary for daughter cell
development. Approximately 85% of parasite phospholipids consist of phosphatidylcholine
(PC) and phosphatidylethanolamine (PE) synthesized by the parasite through the de
novo Kennedy pathways. In the pathway of phosphatidylcholine biosynthesis, the second
step catalyzed by CTP:phosphocholine cytidylyltransferase [EC 2.7.7.15] is rate limiting
and appears essential for the parasite survival at its blood stage [2-3]. We are focused
on the structural characterization of this enzyme, the identification of effectors
by fragment-based drug design approach (FBDD) and then their optimization to eventually
design a lead. The first reported crystal structure of the catalytic domain of the
enzyme target (PfCCT) has been solved at resolution 2.2 Å, 3 enzyme-substrates complexes
(CMP-, phosphocholine- and choline-bound forms) at resolutions 1.9-2 Å and an enzyme-product
(CDP-choline) complex structure at resolution 2.4 Å that give detailed images of binding
pocket, demonstrate conformational changes between apo- and holo-protein forms and
provide the information about the mechanism of the catalytic reaction at atomic level.
The FBDD method uses a library of small molecules (fragments) with molecular weight
that does not exceed 300 Da to explore target binding sites. Although fragments often
have too low affinities to evoke a biological response, their probability of binding
is high because they are small enough to prevent unfavorable interactions with target
protein-binding sites. Moreover, they represent more attractive and synthetically
tractable starting points for medicinal chemistry compared to more complex compounds.
As the affinity is low, fragment screening usually depends on detecting binding rather
than inhibition. Screenings of a fragment library (300 molecules) has been performed
by fluorescence-based thermal shift assay and Nuclear Magnetic Resonance Saturation
Transfer Difference (NMR STD) [4]. This combination of techniques identified so far
4 fragment hits that are currently evaluated for their binding modes and affinities.
Co-crystallization of the protein-fragments complexes is carrying out to provide accurate
information on the molecular interactions. Topology of interactions will be used to
rationally monitor every iterative round of the optimization process allowing subsequent
rational design.
[1] World Health organization, World Malaria Report (WHO Press, Geneva, Switzerland),
http://www.who.int/malaria/publications/world_malaria_report_2014/wmr-2014-no-profiles.pdf?ua=1
[2] Dechamps S, et al., Mol. Biochem. Parasitol., 173:69-80, 2010;
[3] Alberge B, et al., Biochem. J., 425:149-158, 2009;
[4] Mayer and Meyer, Angewand Chem. Int. Ed. 38:1784-8,1999
PB-061
14-3-3 proteins as a scaffold for small-molecule controlled signaling platforms
Anniek Den Hamer1, Lenne Lemmens1, Tom de Greef1, Christian Ottmann1, Maarten Merkx1,
Luc Brunsveld1
1Eindhoven University of Technology
Protein scaffolds play a crucial role in signaling pathways by generating signal specificity
and increasing signal efficiency and amplitude. Engineered protein scaffolds can be
used as key regulators for signal transduction in artificial signal transduction cascades
where they can regulate in- and output of the network. In this research a 14-3-3 protein
scaffold is developed which induces dimerization of proteins mediated by the small
molecule stabilizer fusicoccin. As proof of principle caspase 9 is used to constitute
proximity induced dimerization. Dimerization of caspase 9 leads to its activation
and consecutively initiates the caspase cascade involved in the programmed cell death
pathway. Caspase 9 does not naturally bind to 14-3-3 proteins, therefore the caspase
9 monomer is conjugated to a 14-3-3 binding motif which is known to bind into the
binding grooves of a 14-3-3 dimer. This interaction can be stabilized by the small
molecule fusicoccin. We showed that upon addition of small molecule fusiccocin caspase
dimerization is induced, resulting in caspase activity which is measured using a synthetic
caspase substrate. Moreover the biphasic effect of the 14-3-3 scaffold could be proven.
Additionally, the activated caspase 9 is also able to cleave its natural substrate
caspase 3, downstream in the caspase cascade. These results indicate that the 14-3-3
platform is a versatile small molecule induced dimerization platform which can be
used as tool for engineering of a synthetic signaling network.
PB-062
The G308E variant of the apoptosis inducing factor, responsible of a rare encephalopathy,
is hampered in NAD+/H binding
Luca Sorrentino1, Laura Rigamonti1, Mirvan Krasniqi1, Alessandra Calogero1, Vittorio
Pandini1, Maria Antonietta Vanoni1, Alessandro Aliverti1
1Department of Biosciences, Università degli Studi di Milano
The apoptosis inducing factor (AIF) is a highly conserved mitochondrial flavoprotein
known to play two opposite roles in eukaryotic cells: in mitochondria it is required
for efficient oxidative phosphorylation (OXPHOS), while, when released into the cytoplasm,
it triggers caspase-independent apoptosis (1). The mechanism of AIF-induced apoptosis
was extensively investigated, whereas its mitochondrial role is poorly understood.
There are many evidences of AIF importance for mitochondrial correct morphology and
functions and recently the discovery of its direct interaction with CHCHD4, a key
regulator of respiratory complexes subunits import and folding in mitochondria, was
reported (2). A unique feature of AIF, probably pivotal for its vital function, is
the ability to form a tight, air-stable charge-transfer (CT) complex with NAD+ and
undergo dimerization. Although some aspects of AIF interaction with NAD+/H have been
analyzed, its precise mechanism is not fully understood. We investigated the effect
of the pathogenic G308E replacement, associated with OXPHOS defect and neurodegeneration
(3), to understand how it could alter AIF properties at the molecular level. To do
so, we analysed how the wild type and the G307E forms of murine AIF interact with
NAD+/H and nicotinamide mononucleotide (NMN+/H), finding that the pathogenic replacement
resulted in a dramatic and specific decrease of the rate for CT complex formation
and consequent protein dimerization only in the case of the physiological ligand.
Our results demonstrate that the adenylate moiety of NAD+/H is crucial for the ligand
binding process and that the G307E replacement causes an alteration of the adenylate-binding
site of AIF that drastically decreases the affinity for and the association rate of
the ligand. In addition, we shed new light on the mechanism of the dimerization process,
demonstrating that FAD reduction rather than NAD+/H binding initiates the conformational
rearrangement of AIF that leads to quaternary structure transitions. Taken together,
our results contribute to define how AIF works at the molecular level in binding NAD+/H
and undergoing dimerization and also point out that the G308E replacement, responsible
of a rare neurodegenerative disease, has the selective effect of slowing down the
formation of AIF dimeric CT complex.
1. Sevrioukova (2011) Antioxid Redox Signal, 14: 2545-2579
2. Hangen et al (2015) Molecular Cell, 58: 1-14 3. Berger et al (2011) Mol Genet Metab,
104: 517-520
PB-063
Understanding the mechanism of action of human MICAL1, a multidomain flavoenzyme controlling
cytoskeleton dynamics
Teresa Vitali1, Gabriella Tedeschi2, Simona Nonnis2, Maria Antonietta Vanoni1
1Dipartimento di Bioscienze, Università degli Studi di Milano, 2Dipartimento di Scienze
Veterinarie e Sanità Pubblica, Università degli Studi di
MICAL, from the Molecule Interacting with CasL, indicates a family of conserved cytoplasmic
multidomain proteins that catalyze a NADPH-dependent F-actin depolymerization activity
through their essential N-terminal FAD-containing monooxygenase-like domain (MO) in
response to semaphorin signaling [1]. This domain is followed by calponin homology
(CH) and LIM domains, proline- and glutamate-rich regions and a C-terminal coiled-coil
motif that mediate the interaction with various proteins (e.g: CRMP, CasL, Plexin,
G proteins, NDR) [1]. To contribute to establish the catalytic properties of MICAL
MO and their modulation by the additional domains and by the interacting proteins,
we have produced and are characterizing the human MICAL1 (MICAL-FL) and forms containing
the MO [2], MO-CH and MO-CH-LIM domains. All MICAL forms contain stoichiometric amounts
of FAD in the MO domain and 2 Zn++ ions in the LIM domain. MICAL-MO catalyzes a NADPH
oxidase (H2O2-producing) activity. The CH, LIM and C-terminal domains lower its catalytic
efficiency (kcat/Km, NADPH) mainly due to an increase of Km for NADPH. The kcat is
similar for all forms excepted for MICAL-FL where a 7-fold drop is observed, in agreement
with the proposed autoinhibitory function of the C-terminal domain [3]. The pH dependence
of the kinetic parameters of MO, MOCH and MOCHLIM is complex suggesting that it does
not reflect the ionization state of individual groups, but rather the overall protein
charge. MICAL-MO, -MOCH and -MOCHLIM catalyze a NADPH-dependent F-actin depolymerization
with a similar apparent Km for actin. F-actin (but not G-actin) stimulates the rate
of NADPH oxidation by increasing kcat and lowering KNADPH. The extent of NADPH oxidation
exceeds total F-actin which is in contrast with the proposal of specific modification
of actin Met44 or Met46 reported for Drosophila and mouse MOCH [4-5], but it suggests
that F-actin stimulates the NADPH oxidase activity or a case of substrate recycling.
Accordingly, with hMICAL MO and MOCH several actin residues are oxidized beside Met44
and Met46. Thus, the CH and LIM domains do not seem to be important for the MICAL-actin
interaction and actin modification may be mediated by in situ H2O2 production. In
HEK293T and COS-7 cells mouse collapsin response mediator protein-1 (mCRMP1) interacts
with MICAL1 inhibiting H2O2 production [3], suggesting that CRMP1 could be a hydroxylatable
substrate of MICAL-MO. We have produced the same mCRMP1 form (8-525 aa) and we have
shown that under conditions that limit non specific interactions a mild stimulation
(up to 20%) of NADPH oxidation is observed. F-actin reversed the effect of mCRMP1
suggesting their competition for MICAL. These results suggest that CRMP1, a major
microtubules regulator, is not the substrate of the MO domain, but actin and microtubules
cytoskeleton components may be linked through the formation of CRMP-MICAL complex
in response to semaphorin-plexin signaling. Experiments are in progress to complete
the characterization of MOCHLIM and full length MICAL forms.
1. Vanoni, M.A. et al., (2013).Int J Mol Sci 14, 6920-6959.
2. Zucchini, D. et al., (2011). Arch Biochem Biophys 515, 1-13.
3. Schmidt, E.F. et al., (2008). Neurosci, 28, 2287-2297.
4. Hung, R.J. et al., (2011). Science 334, 1710-1713.
5. Lee, B.C. et al., (2013). Mol Cell 51, 397-404.
PB-064
Protective function of enhanced green fluorescent protein against reactive oxygen
species photo-sensitized by N-doped nanoTiO2
Beata Wielgus-Kutrowska1, Joanna Krasowska1, Agnieszka Bzowska1, László Forró2, Andrzej
Sienkiewicz3
1Department of Biophysics, Institute of Experimental Physics, Warsaw University, 2Laboratory
of Physics of Complex Matter (LPMC), 3ADSresonances
Green fluorescent protein (GFP), owing to its genetically encoded strong fluorescence,
has become one of the most important tools in modern biology [1]. Enhanced GFP (EGFP,
F64L/S65T-GFP), frequently used variants of this protein, is thermodynamically more
stable and ∼35-times brighter than GFP [2]. Due to the improved fluorescent properties,
EGFP is commonly used as a fluorescent intracellular marker in bio-imaging in vitro
and in vivo. Despite sustained interest of the scientific community and numerous practical
applications, the actual biological role of GFP remains elusive. Recent reports put
forward a hypothesis of antioxidant and photo-protective functions of GFP [3]. In
this study, we focused on the photo-protective role of EGFP against reactive oxygen
species (ROS) photo-generated by visible light in water suspensions of nano-particular
nitrogen-doped titanium oxide (N-doped nano-TiO2), that is in the system: ‘N-doped
nano-TiO2)/visible light’. N-doped nano-TiO2 (Sumitomo TP-S201) was chosen as a photo-catalyst,
since it is widely accepted that nitrogen doping enhances visible light photoactivity
of TiO2. 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), a paramagnetic water-soluble
compound, belonging to the nitroxide class o superoxide dismutase (SOD) mimetics,
was used as a target for photo-generated ROS. A solar simulator, with the flux output
intensity of ∼1 kW/m2, was used as a visible light source. Electron spin resonance
(ESR) was employed to monitor the changes in the paramagnetic signal of TEMPOL exposed
to the action of ROS in the absence and presence of EGFP. In the absence of EGFP and
after 50 min of illumination, due to a combined action of superoxide (O2•-) and hydroxyl
(OH•) radicals generated by the system ‘N-doped nano-TiO2)/visible light, the ESR
signal of 100 uM TEMPOL decayed by ∼20%. Moreover, the growth of a new signal, interpreted
as 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPONE), resulting from the attack
of OH•radicals on TEMPOL, was also observed. In contrast, in the presence of EGFP
(7.5 uM), the ROS-induced decay of the ESR signal of TEMPOL was markedly smaller,
not exceeding 5%. Concomitantly, the growth of the ESR signal of TEMPONE was also
partially inhibited (∼30% smaller amplitude), as compared to the process performed
in the absence of EGFP. In summary, our results point to a significant inhibition
of the photo-decomposition of TEMPOL in the presence of EGFP and support the hypothesis
of the protective role of this fluorescent protein against ROS generated by the system
‘N-doped nano-TiO2)/visible light’.
Acknowledgements:
This work was supported by BST 170000/BF (University of Warsaw).
References:
[1] T.D. Craggs, Chem. Soc. Rev. 38, 2856-2875, 2009.
[2] R. Y. Tsien, Annu. Rev. Biochem. 67, 509-644, 1998.
[3] F. Bou-Abdallah, N. D. Chasteen, M.P. Lesser, Biochim. Biophys. Acta 1760, 1690–1695,
2006.
PB-065
Selective Recognition and Assembly in Protein-Small molecule Interactions
Aishling M. Doolan1, Maike C. Jürgens1, Amir R. Khan2, Peter B. Crowley1
1School of Chemistry, National University of Ireland Galway, 2School of Biochemistry
and Immunology, Trinity College Dublin
By studying a variety of anionic ligands and their interactions with cationic cytochrome
c, we are building knowledge of protein recognition geared towards regulating activity.
In previous work it was shown that p-sulfonatocalix[4]arene selectively binds to,
and encapsulates, three Lysine side chains on cytochrome c 1. Here, the binding of
two small molecule ligands to cytochrome c was investigated. NMR spectroscopy was
used and in one case, a crystal structure of the complex was obtained (Fig 1). The
calixarene bound to cytochrome c, reveals a crystal packing assembly that suggests
it is a key mediator of crystal formation. NMR data analysis indicates the calixarene’s
binding site on cytochrome c. The pillararene, a relatively new class of compound,
is a symmetrical arrangement with a π-rich cavity 2, related structurally to calixarenes.
This suggests good host-guest complexation properties. Previously, the carboxylatopillararene
showed selective binding to Arginine, Lysine and Histidine 2. With this ligand, an
interaction with cytochrome c was observed and a complex formed. Additionally, biphasic
binding behaviour was observed through analysis of the chemical shift perturbations.
This may indicate more than one binding event taking place. The data from these studies
indicate that recognition is occurring and again that Lysine side chains play an essential
role.
1. R. E. McGovern, H. Fernandes, A. R. Khan, N. P. Power and P. B. Crowley, Nature
Chemistry, 2012, 4, 527-533.
2. C. Li, J. Ma, L. Zhao, Y. Zhang, Y. Yu, X. Shu, J. Li and X. Jia, Chemical Communications,
2013, 49, 1924-1926.
PB-066
PB-066 Macromolecular crowding modulates enzyme catalysis
Annelise Gorensek1, Luis Acosta1, Gary Pielak1,2,3
1Department of Chemistry, University of North Carolina, 2Department of Biochemistry
and Biophysics, University of North Carolina, 3Lineberger Comprehensive Cancer Center,
University of North Carolina
The enzyme dihydrofolate reductase (DHFR) is necessary for the growth and development
of all organisms. 1 The structure and function of Escherichia coli DHFR have been
characterized in buffer. However, DHFR exists in living cells, where the protein concentration
can exceed 300 g/L. 2 We know that weak, non-specific chemical interactions with cytosolic
proteins alter protein conformation and dynamics,3,4 both of which are expected to
influence DHFR catalysis. Investigators have examined steady-state enzyme kinetics
under crowded conditions, but conclusions can be conflicting. 5,6 Here, the effects
of crowding on E. coli DHFR catalysis are assessed through specific activity measurements
in solutions of synthetic polymers. These kinetics studies are complemented by in-cell
and in vitro 19F NMR data from fluorinated tryptophan residues. Preliminary results
suggest that the effects of polymeric crowders on DHFR activity are non-monotonic,
which may arise from the polymer’s transition from the dilute to semi-dilute regime.
The data suggest that synthetic polymers are not a valid representation of the cellular
interior.
(1) Schnell, J. R.; Dyson, H. J.; Wright, P. E. Annu. Rev. Biophys. Biomol. Struct.
2004, 33, 119.
(2) Zimmerman, S. B.; Trach, S. O. J. Mol. Biol. 1991, 222 (3), 599.
(3) Smith, A. E.; Zhou, L. Z.; Pielak, G. J. Protein Sci. 2015, 24, n/a.
(4) Sarkar, M.; Li, C.; Pielak, G. J. Biophys. Rev. 2013, 5 (2), 187.
(5) Vöpel, T.; Makhatadze, G. I. PLoS One 2012, 7
(6), e39418. (6) Pozdnyakova, I.; Wittung-Stafshede, P. Biochim. Biophys. Acta 2010,
1804 (4), 740.
PB-067
Biophysical and biochemical characterization of Arabidopsis thaliana Calmodulin-like
protein CML14
Rosario Vallone1, Valentina La Verde1, Mariapina D’Onofrio1, Alessandra Astegno1,
Paola Dominici1
1Biotechnology Department, University of Verona
Calcium (Ca2+) is one of the most important second messengers in eukaryotes. Ca2+
binding proteins can be subdivided into two categories: “Ca2+ buffers” that modulate
Ca2+ ion concentrations in cells, and “Ca2+ sensors” that decode Ca2+ signals in a
wide array of physiological processes in response to external stimuli. Calmodulin
(CaM) is the prototypical example of Ca2+ sensor proteins in both animals and plants.
In addition to conserved CaM, plants possess a unique family of 50 CaM-like proteins
(CMLs). Many of these CMLs still remain uncharacterized and the investigation of their
biochemical and biophysical properties will provide insight into Ca2+ signalling in
plants. Herein, a detailed characterization of Arabidopsis thaliana CML14 is reported.
CML14 is a protein of 148 amino acids with a theoretical molecular weight of 16,579
Da and 50% amino acid sequence identity with AtCaM2. CML14 is predicted to have one
functional Ca2+ binding site despite the presence of three EF-hand motifs (Prosite).
We overexpressed CML14 in E. coli and analyzed its biochemical and biophysical characteristics,
i.e. calcium affinity and stoichiometry and eventual changes in conformation, thermal
stability and proteolytic susceptibility upon Ca2+ binding. Isothermal titration calorimetry
(ITC) and nuclear magnetic resonance (NMR) spectroscopy identified one Ca2+ binding
site in CML14 and showed that Ca2+ and Mg2+ compete for the same binding site. The
Kd values determined by ITC established that CML14 has higher affinity for Ca2+ than
for Mg2+. Our data were consistent with the sequence based prediction of one functional
calcium binding site. Differential scanning calorimetry (DSC) showed that Ca2+ and
Mg2+ have the same stabilizing effects on protein folding. Apo-CML14 undergoes two
thermal unfolding transitions, but in the presence of Ca2+ or Mg2+ only one unfolding
event at an intermediate temperature occurs. Limited proteolysis experiments showed
that Ca2+ binding affords protection against CML14 digestion by trypsin. Surprisingly,
CML14 exhibits very few conformational changes upon calcium binding, which were evaluated
by ANS fluorescence and Stokes radius measurements in the apo- and Ca2+ bound-forms.
These results suggest that CML14 does not show the characteristics of a classical
Ca2+ sensor protein. To better understand the physiological role of CML14 in plants,
in vivo analysis will be performed.
PB-068
FBP17 controls the hepatocyte morphology through Rho signaling
Jun Zhang1, Mingming Ling1, Qianying Zhang1, Yunhong Wang1, Deqiang Wang2
1The Department of Cell Biology and Genetics, 2Key Laboratory of Molecular Biology
on Infectious Disease
The formin-binding protein 17 (FBP17) widely expressed in eukaryotic cells was previously
identified to play a role in morphological maintenance in hepatocyte, but the molecular
mechanism keeps still unclear so far. In the present investigation, it was found that
Rho family proteins CDC42/RAC1 signaling was involved in the morphological regulation
controlled by FBP17. Knockdown of endogenous FBP17 expression with RNAi technique
or dominant negative mutant of FBP17 could trigger the cell morphological remodeling
from the epithelioid to fibroid following the significant down-regulation of CDC42/RAC1
activities and dephosphorylation of paxillin. While the Rho protein specific activator
could restore the CDC42/RAC1 activities, and in turn abrogated the silence effect.
Overexpression of wild type FBP17 could not result in any of the morphological transition.
Furthermore, withdrawal of the silence could induce morphological recovery when the
FBP17 expression, CDC42/RAC1 activities and paxillin phosphorylation were restored
to the normal level. The experimental evidences strongly indicated that FBP17 was
implicated in morphological control probably via Rho signaling pathway in hepatocyte.
Key words: FBP17; Rho signaling; paxillin; morphological control; hepatocyte
This work was supported by a grant from National Natural Science Foundation of China
(NSFC, no. 20803098)
PB-069
Energetics of proton transport in Cytochrome c oxidase: Investigation of proton entry
in the K-channel of Paracoccus denitrificans
Jovan Dragelj1, Anna-Lena Woelke1, Ulrike Alexiev2, Ernst-Walter Knapp1
1Fachbereich Biologie, Chemie, Pharmazie/Institute of Chemistry and Biochemistry,
2Fachbereich Physik/Department of Physics
Cytochrome c oxidase (CcO) is the final enzyme in the respiratory chain of mitochondria
but also an integral part of the metabolism of many types of bacteria. In a complex,
stepwise redox-reaction, CcO catalyzes the reduction of molecular oxygen to water
and utilizes the resulting free energy to pump protons across the membrane thereby
creating an electrochemical gradient [1,2]. To investigate proton pumping spectroscopically
it is possible to label the entrance of the proton entrance channel with fluorescein,
a pH sensitive dye, which allows determining time resolved local changes in proton
concentration at the cytoplasmic CcO surface and related properties. It has already
been shown that the redox state of copper and heme centers affects such properties
at the cytoplasmic surface. [3] This study is a theoretical approach to investigate
changes of pKA values of the fluorescein label at the entrance of the K-channel for
different protonation pattern in both oxidized and reduced CcO by performing molecular
dynamics (MD) simulations. Further work is based on calculations of pKA values of
the fluorescein using software Karlsberg+[4,5].
Fig 1.
Entrance of the K-channel of cytochrome c oxidase with attached fluorescein.
1. Brzezinski, P. and R.B. Gennis, J Bioenerg Biomembr, 2008. 40(5): p. 521-31.
2. Kaila, V.R., M.I. Verkhovsky, and M. Wikstrom, Chem Rev, 2010. 110(12): p. 7062-81.
3. Kirchberg, K., Hartmut M., and Ulrike A. Biochimica et Biophysica Acta (BBA)-Bioenergetics
1827, no. 3 (2013): 276-284.
4. Rabenstein, B. and E.W. Knapp, Biophys J, 2001. 80(3): p. 1141-50.
5. Kieseritzky, G. and E.W. Knapp, Proteins, 2008. 71(3): p. 1335-48.
PB-070
Efficient Methods in the Production of Unnatural Amino Acid Containing Proteins
Christopher Walters1, Solongo Batjargal1, Anne Wagner1, E. James Petersson1
1University of Pennsylvania
Methods for genetically and synthetically manipulating protein structure enable a
greater flexibility in the study of protein function. We have shown that using inteins
as traceless, cleavable purification tags enables the separation of full length unnatural
amino acid (Uaa) containing proteins from their corresponding truncation products.
This method has been used to incorporate Uaas in previously unattainable positions
in a variety of proteins using a myriad of Uaas, inteins, and purification tags. In
other applications, we have used E. coli aminoacyl transferase (AaT) to selectively
modify the N-termini of proteins with Uaas in denaturing conditions and conditions
that maintain folding. Applications of particular interest include overcoming the
need for an N-terminal Cys residue in expressed protein ligation, transfer of reactive
handles for “click” chemistry labeling of proteins, and transfer of fluorogenic molecules
for photophysical experiments. We have found that AaT can transfer protected cysteine,
homocysteine, and selenocysteine to expressed proteins. After ligation, these residues
can be converted to Met or Ala, making the ligation traceless. We continue to develop
variants of AaT to broaden the substrate scope of both its transferred substrate and
N-terminal recognition element. In addition, expressed protein ligation is being used
to incorporate backbone modifications, such as the thioamide, into various positions
in the protein calmodulin to determine how these modifications can impact the structure
and function of an ordered protein. In general, by working at the interface of several
protein modification technologies, we have made beneficial discoveries that might
be missed by more focused approaches.
PB-071
Function and modularity of CW_7 motives in the C-terminal region of the endolysin
Cpl-7 encoded by the Cp7 pneumococcal bacteriophage
Manuel Iglesias-Bexiga1,2, Noelia Bernardo-García3, Rubén Martínez-Buey4, Noemí Bustamante1,2,
Guadalupe García1,2, Marta Bruix1, Juan Hermoso3, Margarita Menéndez1,2
1Dept. of Biological Physical-Chemistry, IQFR-CSIC, 2Ciber of Respiratory Diseases
(CIBERES), 3Department of Crystallography and Structural Biology, IQFR-CSIC, 4University
of Salamanca
Bacteriophage lytic murein-hydrolases have been proposed as enzybiotics, an efficient
way to fight bacterial infections. However, the use of these enzymes is normally restricted
to Gram-positive bacteria since the outer membrane of the Gram-negative bacteria hampers
the access of the hydrolases to the peptidoglycan substrates. All the murein hydrolases
reported in the pneumococcal system, both from host or phage origin, depend on the
aminoalcohol choline to be fully active. There is only a unique exception to this
rule, the Cpl-7 lysozyme. This hydrolase is encoded by the lytic pneumococcal phage
Cp-7 and, instead of the common cell wall binding module (CWBM) that recognizes choline,
Cpl-7 harbors a completely different cell wall binding structure. Recent studies have
revealed that reducing the net charge of the CWBM, from −14.9 to +3.0, leads to an
improvement in the antibacterial activity of Cpl-7 (1). The CWBM of Cpl-7 is composed
by three identical repeats of 48 amino acids, the CW_7 motives, and it folds both
in the presence and in the absence of the N-terminal catalytic module (2). This module
shows the capacity of recognize the GlcNAc-MurNAc-L-Ala-D-isoGln muropeptide (GMDP),
structurally related with the peptidoglycan basic unit (3). Here, we report the high
resolution structure of the cell wall binding module of the Cpl-7 endolysin. Each
CW_7 repeat is composed of a bundle of three α-helices with a highly negative electrostatic
charge at the surface. The strong inter-repeat interactions and the high ionic strength
used in the crystallization conditions allow them overcoming the electrostatic repulsions
inducing a closed-packed structure with a three-fold symmetry. The module dimensions
(49 x 38 x 34 Å) and the repeat arrangement in the crystal structure are inconsistent
with the GMDP binding characterization, the activity displayed by Cpl-7 truncated
variants with one or two CW_7 repeats, or the experimental determined hydrodynamic
properties. Using the small angle X-ray scattering (SAXS) technique and the ATSAS
computational platform (4), a different arrangement of the CW_7 repeats is envisaged
in solution (Fig. 1), whose rather opened structure (70 x 44 x 46 Å) is consistent
with the experimental data. Additionally, employing the SAXS-based structure and the
honeycomb structure proposed for the peptidoglycan, a model, where each CW_7 repeat
of the cell wall binding module fit in adjacent glycan chains, has been derived.
Fig. 1.
Experimental SAXS data of C-terminal domain of Cpl-7 (black dots) and the theoretical
scattering profiles calculated by CRYSOL (4) from the x-ray structure (orange line
and cartoon) and from the ab initio SAXS model (grey line and beads model).
1. Bustamante, N. et al., Cpl-7 a lysozyme encoded by a pneumococcal bacteriophage
with a novel cell wall-binding motif. 2010. J. Biol. Chem. 285, 33184-33196.
2. Díez-Martínez, R. et al., Improving the lethal effect of Cpl-7, a pneumococcal
lysozyme with broad bactericidal activity, by inverting the net charge of its cell
wall-binding module. 2013. Antimicrob Agents Chemother 57, 5355-5365.
3. Bustamante, N. et al., The Cpl-7 endolysin from Cp-7 pneumococcal bacteriophage:
thermal stability and cell wall-targeting specifity. 2012. Plos One 7, (10):e46654.
4. Petoukhov, M.V. et al., New developments in the ATSAS program package for small-angle
scattering data analysis.2012. J. Appl. Cryst. 45, 342-350.
PB-072
Utilizing computational and experimental chemistry to characterize the functions of
Structural Genomics proteins in the Crotonase Superfamily
Caitlyn Mills1, Pengcheng Yin1, Penny Beuning1, Mary Jo Ondrechen1
1Northeastern University
In 2000, the Protein Structure Initiative (PSI) was started as to determine three-dimensional
structures of proteins within every family. Once solved, structures are deposited
into the Protein Data Bank (PDB) and termed Structural Genomics (SG) proteins. As
of June 2015, there are over 13,300 SG proteins deposited in the PDB and most of them
are of unknown or uncertain biochemical function. In addition, many of these SG proteins
have a putative functional assignment based on their sequence and structural similarities
with proteins of known function; such comparisons can be made against large databases
using programs such as BLAST or Dali. However, these putative functional assignments
are often incorrect. This project analyzes members of the Crotonase Superfamily (CS).
The CS consists of five diverse functional subgroups that are well characterized structurally
and functionally, representing different types of reactivity, including hydrolase,
isomerase, hydratase, and dehalogenase activities. This superfamily also contains
at least 70 SG proteins, so it is ideal to test predictions of protein function. Our
approach is based on local structure matching at the computationally predicted active
site. First, Partial Order Optimum Likelihood (POOL) is used to predict the functionally
important residues of each SG protein and of the proteins of known function in the
superfamily. Next, Structurally Aligned Local Sites of Activity (SALSA) is used to
align the predicted catalytic residues of the well-characterized members in the superfamily.
From this analysis we generate chemical signatures for each functional subgroup and
compare them to the sets of catalytic residues predicted for the SG proteins. We demonstrate
based on these computational methods that the majority of the putative annotations
in the CS superfamily are likely incorrect. Currently, biochemical assays are being
used to test these predictions. Preliminary biochemical results show that one SG protein,
Thermus thermophilus Q5SLS5_THET8, classified as a probable enoyl-CoA hydratase, possesses
hydrolase activity as predicted by our methods. The outcomes of this project will
be to successfully classify the biochemical functions of SG proteins based on their
local structure at the predicted active sites and to provide a conceptual framework
for the functional classification of the remaining SG proteins within the PDB.
This work is supported by NSF-CHE-1305655.
PB-073
Directly observing the synergistic dynamics in F-actin and microtubule assembly
Jun Zhang1, Deqiang Wang2
1The Department of Cell Biology and Genetics, 2Key Laboratory of Molecular Biology
on Infectious Disease
Although important in cellular activities, little attention was paid to the synergistic
effects of actin and microtubule cytoskeleton assembly. With the time-lapse atomic
force microscope (TL-AFM), we directly observed the large-scale dynamic structure
of actin filaments formed in the presence or absence of microtubulin in solution.
In absence of microtubulin, the G-actin could be polymerized into ordered filamentous
structures with different diameter from the slimmest filament of single F-actin to
giant filament in tree-like branched aggregates. The polymerized actin filaments,
to which our most intense attention was attracted, was discretely arranged and showed
obvious polymorphism in structures completely distinct from those in the presence
of microtubulin. The supra-molecular complex structures of the latter were mainly
composed of single F-actin and/or multifilaments clearly consisting of several single
F-actin and regularly cross-linked with the assembled microtubular bundles. The experimental
results demonstrated that the F-actin dynamics could be coordinated by microtubule
assembly. Further analyses implied that the interactions between F-actin and microtubule
could prevent the emergence of structural polymorphism of F-actin alone, and give
rise to organization of specific complex structures instead. It was suggested that
dynamic synergy between the F-actin and microtubule would be implicated in living
cells.
Key words:
TL-AFM; actin; microtubule; assembly dynamics; synergy
PB-074
Bivalent phosphonate inhibitors for extracellular 14-3-3 protein targets
Jeroen Briels1, Maria Bartel1, Elvan Yilmaz2, Philipp Thiel3, Markus Kaiser2, Christian
Ottmann1
1Laboratory of Chemical Biology, Eindhoven University of Technology, 2Centre for Medical
Biotechnology, University of Duisburg-Essen, 3Department of Computer Science, University
of Tübingen
The adaptor protein 14-3-3 is found in a diverse range of pathologically relevant
protein-protein interactions (PPIs). As 14-3-3 is a hub protein with very diverse
interactions, it is able to influence the intracellular localization of their binding
partners and they are key regulators of signal transduction processes as well as regulators
of cell cycle functions.Nevertheless, there are only few examples of 14-3-3 acting
extracellularly. One of the extracellular targets for 14-3-3 is Aminopeptidase N (APN).
APN is an extracellular trans-membrane enzyme that acts as a receptor for 14-3-3.
Binding to APN, 14-3-3 excreted by keratinocytes can upregulate the excretion of matrix
metalloproteinase-1 (MMP1) in fibroblasts. MMP1, by breaking down collagens, is key
in the remodeling of the extracellular matrix. Modulation of the 14-3-3/APN interaction
thereby may play a crucial role in the fundamental understanding and ultimately treatment
of wound healing, respiratory diseases and tumor growth.
In the eukaryotic cell, the 14-3-3 dimer operates as an adapter platform for binding
partners. A wide range of classes of (small) molecules, natural products and peptides
has been used to modulate the PPIs, providing either stabilization or inhibition of
the interactions of 14-3-3 with its binding partner. Binding partner fragments or
peptides are known to bind to the 14-3-3 binding groove via arecognition motif containing
a phosphorylated serine or threonine.
Making use of the dimeric structure of 14-3-3, novel small-molecule inhibitors may
be tethered to exploit the bivalent effect. From a large virtual screening and experimental
validation, a scaffold containing a phenyl phosphonic moiety was identified, showing
inhibitory properties for 14-3-3 PPIs. Potent derivatives of this scaffold were bridged
by polyethylene glycol (PEG) linkers of varying lengths, thereby facilitating the
compound to reach both binding sites of the 14-3-3 dimer and concurrently increasing
the compound’s solubility in aqueous solution. Similar bivalent inhibitors have been
proven to synergistically increase their efficacy.
Biophysical evaluation by means of fluorescence polarization (FP) inhibition competition
assays, revealed an increase of the half maximal inhibitory concentration (IC50) from
approximately 81 μM for the monomeric phenyl phosphonate to approximately 1.8 μM for
the bivalent inhibitor with a 60Å linker. This demonstrates a 45-fold increase of
inhibitory effect towards 14-3-3 and its binding partner peptide mimic. Extensive
thermodynamic, kinetic and structural analysis of the interaction is in progress.Phosphonic
moieties have been shown to pass the cell membrane poorly, due to their highly charged
character. By being able to specifically inhibit the extracellular interaction between
14-3-3 and APN, these inhibitors are prevented from interfering with the extensive
intracellular 14-3-3 interactome. Hence, these bivalent phenyl phosphonate inhibitors
provide a promising strategy towards extracellular application.
PB-075
Probing the extremely high metal-to-protein affinity of interprotein zinc hook domain
of Rad50 protein from P. furiosus
Tomasz Kochanczyk1, Michal Nowakowski2, Dominika Wojewska1, Artur Krezel1
1Laboratory of Chemical Biology, Faculty of Biotechnology, University of Wroclaw,
2Laboratory of NMR Spectroscopy, Center of New Technology, University of Warsaw
The Mre11 complex is an oligomeric assembly comprising of dimmers of Mre11 and Rad50
proteins in Archea and additionally Nbs1 subunit present in Eukaryote. It is the central
player in the DNA damage response - a functional network comprising DNA damage sensing,
signal transduction, cell cycle regulation and DNA double strand breaks (DSBs) repair
[1]. Recent structural studies revealed that Rad50 hinge domain is rather a short
kink in the coiled-coil region and adopts unusual dimerization mode by intermolecular
coordination of Zn(II) and formation of so-called zinc hook domain [2]. To date, very
limited structural data on the zinc hook domain have been reported, the only known
structure was resolved for Rad50 homologue from hyperthermophilic archaeon – P. furiosus.
Unusual Zn(II) coordination mode in zinc hook domain raises question of how zinc hook
domain assembles to form interprotein zinc binding site with sufficient stability
to function at low intracellular free Zn(II) concentrations [3]. Our study on minimal
zinc hook domain fragment (14 aa) indicated low femtomolar affinity towards Zn(II)
[4]. Extended zinc hook domain fragment (45 aa) reveals even zeptomolar affinity.
Therefore, our main goal was to probe the thermodynamic and structural effects that
are hidden in the small interprotein interface and are responsible for the dimerization
of the large and critical protein machinery. Probing of those effects was achieved
by detailed biophysical characterizations (including potentiometry, NMR, HDX MS and
CD spectroscopy) of 18 protein fragments of zinc hook domains with a number of point
mutations. We showed that extremely high stability of zinc hook domain from P. furiosus
is achieved by the formation of hydrogen bond network in β-hairpins and interprotein
hydrophobic core.
This work was supported by the National Science Centre, grant: 2014/13/B/NZ1/00935
and Polish Foundation for Science (F1/2010/P/2013).
[1] Hohl, Kochańczyk et al. (2015) Mol. Cell, 57, 479.
[2] Hopfner et al. (2002) Nature 2002, 418.
[3] Kochańczyk, Drozd, Krężel (2015) Metallomics, 7, 244
[4] Kochańczyk, Jakimowicz, Krężel (2013) Chem. Comm. 49, 1312.
PB-076
DNA-directed control of enzyme-inhibitor complex formation: A modular approach to
reversibly switch enzyme activity
Wouter Engelen1, Brian Janssen1, Maarten Merkx1
1Eindhoven University of Technology
DNA-based molecular circuits have become a very attractive tool in molecular imaging,
synthetic biology, molecular diagnostics and biomolecular computing. The highly modular
and predictable nature of Watson-Crick base pairing allows the construction of complex
circuits using a limited set of logic gates and building blocks. However, the lack
of generic approaches to interface DNA-based molecular circuits with protein activity
limits their application in biomedicine and molecular diagnostics. Here we present
a new, highly modular approach to control the activity of a reporter enzyme based
on the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase
and its inhibitor protein BLIP. Both proteins are conjugated to a unique oligonucleotide,
allowing the assembly of the enzyme-inhibitor pair and inhibition of enzyme activity
by the addition of a complementary template strand. Addition of an oligonucleotide
that is complementary to a loop sequence in the template results in the formation
of a rigid dsDNA spacer that disrupts the enzyme-inhibitor complex, restoring enzyme
activity. Using this noncovalent approach allowed easy tuning of the template and
target sequences with only a single set of oligonucleotide-functionalized enzyme and
inhibitor. To show the modularity of the system, a panel of 8 different template sequences
were selected. Only in the presence of their complementary viral DNA sequences restoration
of enzyme activity was observed. In addition to this excellent specificity the system
showed to by higly sensitive towards its target, since the presence of as little as
2 fmol of target resulted in an observable increase in enzyme activity. The use of
a stable and well-characterized enzyme-inhibitor pair, complemented by the modular
design of our reversible DNA-directed protein switch make it an attractive system
to implement in DNA-based molecular circuits.
PB-077
Carboxylic Acids: a versatile classe of carbonic anhydrase inhibitors
Giuseppina De Simone1, Simone Carradori2, Emma Langella1, Simona Maria Monti1, Claudiu
T. Supuran3, Katia D’Ambrosio1
1Istituto di Biostrutture e Bioimmagini-CNR, 2Department of Pharmacy, ‘G. D’Annunzio’
University of Chieti-Pescara, 3Università Degli Studi Di Firenze, NEUROFARBA Department
Several studies demonstrated important roles of human Carbonic anhydrases (hCAs) in
a variety of physiological and pathological processes. Consequently, in recent years
the 12 catalytically active hCA isoforms have become an interesting target for the
design of inhibitors with biomedical applications [1]. Derivatized sulfonamides of
type R-SO2NH2 represent the class of CA inhibitors (CAIs) mostly used and best characterized.
The large number of crystallographic studies so far available on these molecules clarified
the main factors responsible for the binding of the sulfonamide moiety to the CA active
site. 1 In particular, it has been highlighted that even though these molecules generally
behave as very potent CAIs, they do not show selectivity for the different isoforms.
Indeed, the sulfonamide moiety plays a predominant role in the interaction with the
enzyme, while any change in the nature of the R substituent has generally a rather
marginal effect on the enzyme-inhibitor affinity. These characteristics make difficult
the design of sulfonamide derivatives selective for the different CA isoforms. Consequently,
much efforts were dedicated in last years to the development of new inhibitors that,
although presenting lower affinity for the CA active site, would be able to be more
selective toward the different isoforms. Carboxylic acids have been recently investigated
as CAIs, showing that these molecules can adopt different binding modes to the enzyme
active site. In particular, they can coordinate directly to the zinc ion or be anchored
to the zinc-bound water molecule. However, the structural reasons responsible of this
peculiar behavior have not been clarified yet. In a general research project aimed
at providing insights into the binding mode of these molecules to CAs, we have undertaken
the characterization of two carboxylic acids, namely an ortho-substituted benzoic
acid [2] and a saccharine derivative, by means of kinetic, crystallographic and theoretical
studies.
[1] Alterio V., Di Fiore A., D’Ambrosio K., Supuran C.T., De Simone G. Chem. Rev.
2012, 112:4421-68.
[2] D’Ambrosio K., Carradori S., Monti S.M., Buonanno M., Secci D., Vullo D., Supuran
C.T., De Simone G. Chem. Commun. 2015, 51:302-5.
PB-078
Exploring the mechanism of fibril formation using fluorescently labelled human lysozyme
variants
Ana Bernardo Gancedo1
1University of Cambridge
’Exploring the mechanism of fibril formation using fluorescently labelled human lysozyme
variants’ Human lysozyme is a widely characterised protein whose mutational variants
misfold into fibrils that are associated with systemic amyloidosis (1). Although the
process of aggregation for human lysozyme has been well studied, the details of early
events within this process are not fully characterised. Single molecule fluorescence
microscopy has been used to determine the oligomeric distributions present in the
aggregation process of a number of disease-related intrinsic disordered proteins (IDPs)
(2). Recent advances in site-specific labelling of human lysozyme (3) have made this
protein amenable to these single molecule fluorescence studies. We have introduced
Alexa-fluorophores into the I59T variant of human lysozyme and have demonstrated that
the process of in vitro fibril formation is not significantly altered. Using these
fluorophore-labelled proteins we can apply single molecule fluorescence to study the
early aggregation events within this system, allowing us to compare protein aggregation
in a globular protein and with the aggregation process of IDP’s.
1. Dumoulin M, Kumita JR, Dobson CM (2006) Normal and aberrant biological self-assembly:
insights from studies of human lysozyme and its amyloidogenic variants. Acc Chem Res
39: 603–610.
2. Cremades N, Cohen SIA, Deas E, Abramov AY, Chen AY, Orte A, Sandal M, Clarke RW,
Dunne P, Aprile FA, Bertoncini CW, Wood NW, Knowles TP, Dobson CM, and Klenerman D.
(2012) Direct observation of the interconversion of normal and toxic forms of a-synuclein.
Cell 149: 1048–1059
3. Ahn M, De Genst E, Kaminski Schierle GD, Erdelyi M, Kaminski CF, Dobson CM, Kumita
JR (2012) Analysis of the native structure, stability and aggregation of biotinylated
Human Lysozyme. PLos One 7: e50192
PB-079
A new lead compound for the development of Carbonic Anhydrase inhibitors
Anna Di Fiore1, Giuseppina De Simone1, Alessandro Vergara1,2, Marco Caterino2, Vincenzo
Alterio1, Simona M. Monti1, Joanna Ombouma3, Pascal Dumy3, Claudiu T. Supuran4, Jean-Yves
Winum3
1Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16 - 80134 Naples.,
2University of Naples Federico II, Via Cinthia - 80126, Naples., 3Institut des Biomolécules
Max Mousseron-CNRS, Université de Montpellier., 4Università Degli Studi Di Firenze,
NEUROFARBA Department
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which catalyze the reversible
hydration of carbon dioxide to bicarbonate ion and proton. These proteins are present
in prokaryotes and eukaryotes, and are encoded by six evolutionarily unrelated gene
families.[1,2] Human CAs (hCAs) are widely distributed in many tissues and organs.
Since at these sites CAs play a crucial role in various physiological processes, they
have recently become interesting targets for pharmaceutical research. Indeed, several
CA inhibitors (CAIs) incorporating a sulfonamide/sulfamate/sulfamide moieties are
currently clinically used for the treatment or prevention of a multitude of diseases
such as glaucoma, solid tumors, etc.[1] However, these compounds are generally poorly
selective towards the different CA isoforms. For this reason, despite many encouraging
results, new zinc binding groups (ZBGs) are continuously tested in order to advance
upon the identification of isoform selective CAIs.[1] Here, we report the synthesis,
inhibition and structural studies of hydroxylamine-O-sulfonamide, a molecule containing
two ZBGs, namely the sulfonamide and hydroxylamine moieties.[3] The inhibitor action
of this molecule was tested against all catalytically active hCA isoforms, revealing
that it possesses a rather variable behavior against the different human isozymes.
To elucidate the binding mode of hydroxylamine-O-sulfonamide to CA active site, an
original crystallography-assisted Raman spectroscopy approach was utilized. In particular,
the X-ray structures of hydroxylamine-O-sulfonamide in complex with hCA II, the best
characterized hCA isoform, showed that hydroxylamine-O-sulfonamide is in part coordinated
in the classical manner, as all sulfonamides/sulfamates, binding the catalytic zinc
ion through the sulfonamide nitrogen and in part through hydroxylamine moiety in a
side-on fashion, which is an unusual inhibition pattern for this family of enzymes.
This surprising observation was further proved by Raman microspectroscopy experiments.
Altogether our data indicate that the hydroxylamine-O-sulfonamide versatility can
be exploited for drug design purposes for obtaining new effective and selective CAIs.
1. Alterio, V., Di Fiore, A., D’Ambrosio, K., Supuran, C.T., De Simone, G. Chem. Rev.,
2012, 112, 4421.
2. De Simone, G., Di Fiore, A., Capasso C., Supuran, C. T. Bioorg. Med. Chem. Lett.,
2015, 25, 1385.
3. Di Fiore A, Vergara A, Caterino M, Alterio V, Monti SM, Ombouma J, Dumy P, Vullo
D, Supuran CT, Winum JY, De Simone G. Chem. Commun. (Camb.), 2015. In press.
PB-080
Secondary transporter structure and function in synthetic lipid bilayer systems
Heather Findlay1, Sowmya Purushothaman2, Oscar Ces2, Paula Booth1
1Kings College London, 2Imperial College London
Biological membranes are complex environments, where membrane proteins are surrounded
by a bilayer composed of many different types of lipid. The physical properties of
the bilayer influence protein structure, folding and function, while specific interactions
with lipid molecules can also contribute towards the biological activity of some membrane
proteins. Improving understanding of the interactions has resulted in the development
of artificial lipid systems that allow the bilayer properties to be rationally manipulated
in vitro to control protein behaviour. The bacterial transporter LacY is a well known
integral membrane protein from the Major Facilitor Superfamily, responsible for the
proton-driven uptake of D-lactose in E. coli. With a high resolution structure available
and considerable understanding of mechanistic detail, and with observed changes to
both structure and function in different bilayer environments, LacY is a good model
system for examining the behaviour of a major class of membrane proteins in these
lipid systems. Purified LacY has been reconstituted into liposomes and droplet interface
bilayer systems of varying lipid composition and the effect on protein function and
bilayer properties examined.
PB-081
Targeting Abeta oligomers by Trehalose-conjugated peptides: a molecular dynamics study
Emma Langella1, Ida Autiero1, Michele Saviano2
1National Research Council, Institute of Biostructures and Bioimaging, 2National Research
Council, Institute of Crystallography
Targeting Abeta oligomers by Trehalose-conjugated peptides: a molecular dynamics study
Emma Langellaa, Ida Autieroa and Michele Savianob a National Research Council, Institute
of Biostructures and Bioimaging, 80138 Naples, Italy b National Research Council,
Institute of Crystallography, 70126 Bari, Italy Alzheimer’s disease (AD) is currently
one of the most common and devastating forms of dementia correlated with beta-amyloid
peptide (Abeta) accumulation in human brain tissue [1,2]. Inhibiting Abeta self-oligomerization
in brain tissue remains one of the main strategies to prevent or treat this disorder.
As a consequence, in recent years much efforts have been spent in the understanding
of the amyloid fibril growth process and its modulation by putative drug molecules.
An interesting class of compounds able to prevent Abeta fibrillogenesis, is represented
by beta-sheet-breaker (BSB) peptides [3]. Although these molecules are thought to
recognize in a self-complementary manner the Abeta hydrophobic core region, however
their precise mechanism of interaction is still unclear. In this context, we have
studied the structural basis underlying the inhibitory effect of Abeta(1-42) fibrillogenesis
explicated by two promising trehalose-conjugated BSB peptides (Ac-LPFFD-Th (ThCT)
and Th-Succinyl-LPFFD-NH2 (ThNT)) [4] using an all-atom molecular dynamics (MD) approach
[5,6]. The pentameric NMR structure [7] of Abeta(1-42) has been used to model amyloid
protofibril, and the two protofibril ends have been investigated as putative binding
sites. Our simulations suggest that the interaction with the two protofibril ends
occurs through different binding modes. In particular, binding on the odd edge (chain
A) is guided by a well defined hydrophobic cleft, which is common to both ligands
(ThCT and ThNT). Moreover, targeting chain A entails a significant structure destabilization
leading to a partial loss of β structure and is an energetically favoured process,
as assessed by MM/PBSA calculations. A significant contribution of the trehalose moiety
to complexes stabilities emerged from our results. The energetically favoured hydrophobic
cleft detected on chain A could represent a good starting point for the design of
new molecules with improved anti-aggregating features.
[1] Hardy J. and Dennis J.S., Science, 2002, 297, 353-356
[2] Adessi C. and Soto C., Drug Dev Res. 2002, 56, 184-193
[3] Bieler S., Soto C., Curr Drug Target, 2004, 5(6):553-8.
[4] De Bona P., Giuffrida M.L., Caraci F., Copani A., Pignataro B., Attanasio F.,
Cataldo S., Pappalardo G. and Rizzarelli E., J. Pept Scie., 2009, 15, 220-228.
[5] Autiero I, Saviano M, and Langella E. Mol Biosyst. 2013, 8, 2118-24.
[6] Autiero I., Langella E. and Saviano M. Mol. BioSyst., 2013, 9, 2835-41.
[7] Luhrs T., Ritter C., Adrian M., Riek-Loher D., Bohrmann B., Doeli H., Schubert
D. and Riek R., Proc.Natl. Acad. Sci. U. S. A., 2005, 102, 17342–17347.
PB-082
Establishing a tool box for generating designer nucleosomes
Diego Aparicio Pelaz1, Henriette Mahler, Dirk Schwarzer, Wolfgang Fischle
1 University of Tuebingen
The basic structural unit of chromatin is the nucleosome, which is composed of histone
proteins forming a scaffold with about 150 base pairs of DNA wrapped around. Chromatin
compacts eukaryotic genomes and regulates gene activity, which is mediated in part
by posttranslational modifications (PTMs) on the N-terminal tails of the histones.
Uncovering the detailed relationship between histone tail modifications and gene activity
is a major topic of biomedical sciences and general techniques for generating nucleosomes
with defined modification patterns in large numbers would greatly facilitate such
investigations. To this end we are establishing a chemical toolbox for designer chromatin
with defined histone PTM patterns. A protein semysinthesis approach is used that bases
on “ligation-ready nucleosomes” with truncated histone H3 that can be ligated with
the corresponding synthetic histone tail. We resorted to sortase-mediated ligation
as chemoselective ligation method. Here we report our recent developments in establishing
the envisioned chemical toolbox for designer chromatin.
PB-083
Evaluating cation-pi and pi-pi interaction in proteins using various biophysical methods
Jinfeng Shao1, Andy-Mark W.H. Thunnissen1, Jaap Broos1
1Laboratory of Biophysical Chemistry, University of Groningen Nijenborgh
In proteins the aromatic residues phenylalanine (Phe), tyrosine (Tyr), and tryptophan
(Trp) can be involved in aromatic interactions known as cation-pi and pi-pi interactions
(Dougherty 2000). Compared to other non covalent interactions in proteins, like H-bonds,
dipole-dipole, or van der Waals interactions, relatively little is known about the
pi-pi and the cation-pi interactions. The strength of both aromatic interactions is
dependent on the pi-electron density in the aromatic residues. A lowering of electron
density can be created by introducing strong electron-withdrawing substituents like
fluorine atoms in the aromatic ring (Dougherty 2000). In this way a nearly isosteric
change in the aromatic system results in a marked change in electron density. Substitution
with methyl groups is known to slightly increase the electron density. The published
protein labeling method only yields ng quantities of labeled proteins, limiting the
scope to channel proteins and their characterization via patch-clamp. The protein
LmrR is a transcription factor in Lactococcus lactis that regulates the drug-induced
expression of the ABC type multidrug efflux transporter LmrCD (Madoori et al. 2009).
LmrR was crystallized in its drug-free state and in complex with the aromatic substrates
Hoechst 33342 (H33342) and daunomycin (Madoori et al. 2009). Each substrate is sandwiched
between two Trp residues, suggesting pi-pi interactions are operative. To study this
interaction in more detail LmrR was biosynthetically labeled with Trp analogs which
differ in electron density in the indole moiety. This was achieved by co-expressing
LmrR and the Trp-tRNAsynthetase in a Lactococcus lactis Trp auxotroph (Petrovic et
al. 2013), yielding mg quantities of labeled proteins. 5-Fluoro-Trp, 5,6-difluoro-Trp,
and 5-methyl-Trp were incorporated into LmrR successfully with very high efficiencies.
Various biophysical methods including steady state and stopped-flow fluorescence spectroscopy,
surface plasmon resonance, and X-ray crystallography are used to evaluate the binding
interaction. Our results indicate pi-pi interactions play a role in binding of drugs
to LmrR. Recently, we also demonstrated the efficient incorporation of β-(1-azulenyl)-L-alanine
in LmrR (Shao et al. 2015). Give its spectroscopic properties (UV-Vis, fluorescence,
infrared), this blue colored isostere of tryptophan offers great potential as protein
spectroscopic probe under in vitro and in vivo conditions.
Dougherty, D.A. 2000. Unnatural amino acids as probes of protein structure and function.
Current Opinion in Chemical Biology 4:645-652.
Madoori, P.K., Agustiandari, H., Driessen, A.J.M., and Thunnissen, A.M.W.H. 2009.
Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition.
EMBO J. 28:156-166.
Petrovic, D.M., Leenhouts, K., van Roosmalen, M.L., and Broos, J. 2013. An expression
system for the efficient incorporation of an expanded set of tryptophan analogues.
Amino Acids 44:1329-1336.
Shao, J., Korendovych, I.V., and Broos, J. 2015. Biosynthetic incorporation of the
azulene moiety in proteins with high efficiency. Amino Acids 47:213-216.
PB-084
Synthesis and application of chemical probes for histone deacetylases
Julia Sindlinger1, Alexander Dose1, Jan Bierlmeier1, Frank Essmann1, Markus Hartl2,
Iris Finkemeier3, Dirk Schwarzer1
1Interfaculty Institute of Biochemistry, University of Tuebingen, 2Max Planck Institute
of Biochemistry, 3Max Planck Institute for Plant Breeding Research
Histone Deacetylaces (HDACs) and Histone Acetylaces (HATs) are responsible for maintaining
the global acetylation-level of proteins, including histones. Lysine-acetylation influences
gene expression by varying the accessibility of DNA for transcription factors and
other chromatin binding proteins [1]. Alterations in the HAT and HDAC activities and
the resulting changes in gene expression are therefore considered as key factors in
the pathogenesis of cancer and other diseases. Consequential HDACs represent promising
drug targets for cancer therapy. Several potent HDAC inhibitors have been reported
to date and some of them have been approved as drugs [2]. However, the poor accessibility
of recombinant HDACs hampers biochemical research on this important class of enzymes.
We have developed peptide-based probes that allow investigations on endogenous HDACs
in cellular lysates. These probes were used in pull-down experiments to isolate HDAC
complexes and study their substrate selectivity.
[1] Strahl, B.D., T. and Allis, C. D., Nature 403, 41-46 (2000)
[2] Minucci. S. and Pelicci, P. G., Nat. Rev. Cancer 6, 38-51 (2006)
PB-085
Exploring the Substrate Selectivity of Oxygen Sensing Prolyl Hydroxylases
Kerstin Lippl1, Martine Abboud1, Christoph Loenarz2, Christopher Schofield1
1Department of Chemistry, University of Oxford, United Kingdom, 2Department of Chemistry,
University of Nottingham, United Kingdom
The response to low cellular oxygen levels in humans and other animals is induced
by the hypoxia inducible transcription factors (HIFs). These transcription factors
are regulated by hypoxia inducible factor prolyl hydroxylases (PHDs), which act as
‘oxygen sensors’ by hydroxylating HIFs, thus leading to the proteomic degradation
of the transcription factors. Over the last years, there have been multiple reports
that describe additional PHD substrates other than HIFs. Among them are the large
subunit of RNA Pol II, several transcription factors, and components of signalling
pathways. Validating these reports is of major medicinal relevance given that PHD
inhibitors are now in the late stage Phase 3 clinical trials. In order to investigate
the selectivity of PHDs, the reported proteins have been tested as substrates for
hydroxylation by Mass Spectrometry, and as binders or competitors of the PHDs. Initial
work on peptides that contain the putative hydroxylation sites has indicated that
the PHDs are much more selective for their well-established substrate HIF. However,
in ongoing work these initial results are going to be validated on protein level by
co-expressing PHDs with the reported substrates. Additionally, peptides of reported
substrates were screened for their ability to alter the kinetics of HIF-hydroxylation
by PHD2. An inhibitory effect of at least two different peptides on PHD2 was observed,
suggesting that there is an interaction between the prolyl hydroxylase and these peptides.
In order to investigate the mode of binding and inhibition, NMR studies have been
carried out and binding of the two inhibitory peptides on PHD2 has been shown. Altogether,
these results indicate that, although PHDs might be more selective for HIF as a substrate
as it was initially thought, the enzymatic activity of the prolyl hydroxylases is
possibly influenced by a number of other proteins that can directly bind to PHDs.
PB-086
Non-natural aminoacids via the MIO–enzyme toolkit
Alina Filip1, Judith H Bartha-Vári1, Gergely Bánóczy2, László Poppe2, Csaba Paizs1,
Florin-Dan Irimie1
1Biocatalysis and Biotransformation Research Group, Department of Chemistry, UBB,
2Department of Organic Chemistry and Technology
An attractive enzymatic route to enantiomerically pure to the highly valuable α- or
β- aromatic amino acids involves the use of aromatic ammonia lyases (ALs) and aminomutases
(AMs). All these enzymes have in common an auto-catalically formed 5-methylene-3,5-dihydroimidazole-4-one
(MIO) electrophilic prosthetic group, and show high structural and sequence similarities.
The recent advances in improving the functional properties of these enzymes increased
both their biocatalytic and therapeutic applications.
Scheme 1.
Biotransformation with PAL and PAM
We aimed to create a library of recombinant MIO-enzymes consisting of the PALs and
PAMs with large substrate promiscuity in order to provide access to various non-natural
aminoacids through enzymatic ammonia addition and/or ammonia elimination reactions
of the substrate library already available in our researchgroup. The developed complementary
substrate and enzyme library would provide the MIO-enzyme toolkit useful for the synthesis
of nonnatural aminoacids. The synthetic gene of the enzymes (PcPAL, RtPAL, AvPAL,
PaPAM) were cloned into pET19b_J906 expression vector using XhoI and Bpu1102I cloning
sites. The plasmid DNA was transformed to several E.coli host strains (Rosetta, BL21,
Origami 2) in order to optimize the expression yields. The enzymes containing an N-terminal
His10-tag were purified with affinity chromatography, followed by ion-exchange or/and
size-exclusion chromatography, obtaining pure and homogenous proteins, in their tetrameric,
presumably native fold. The enzyme activity and the kinetic parameters of the purified
enzymes was determined towards the natural substrate L-phenylalanine, as well as towards
novel bulkier aromatic substrates (heteroaryl alanines, styryl alanines, biphenylalanines).
Furthermore to enhance their biocatalytic applicability we covalently immobilized
the enzymes to carboxylated single-walled carbon nanotubes (SwCNT COOH) using linkers
with different lengths, and tested the activity and recycling of the immobilized enzyme.
Acknowledgements: AF thanks for the financial support of the Sectorial Operational
Program for Human Resources Development 2007-2013, co-financed by the European Social
Fund, under the project number POSDRU/159/1.5/S/132400 and of the Romanian National
Authority for Scientific Research, CNCS-UEFISCDI,project number PN-II-ID-PCE-2011-3-0775.
LP thanks for financial support from Hungarian OTKA Foundation (NN-103242) and from
the New Hungary Development Plan (TÁMOP-4.2.1/B-09/1/KMR-2010-0002: Development of
quality-oriented and harmonized R+D+I strategy and functional model at BME). The authors
also thank the support from COST Action CM1303 (SysBiocat) and Jody L. McGinness
PB-087
High affinity synthetic antibodies as biological tools
Mateusz Lugowski1, Malgorzata Nocula-Lugowska1, Somnath Mukherjee1, Anthony Kossiakoff1
1Department of Biochemistry and Molecular Biology, The University of Chicago
Antibodies that bind protein antigens are indispensable tools in biochemical research
and modern medicine. Utilizing a phage display selection strategy, we have obtained
synthetic antigen binders (sABs), based on a Fab fragment of IgG, to a wide array
of proteins as distinct as membrane proteins, structural proteins, scaffold proteins
and nuclear targets. Here we demonstrate the applicability of the sABs towards the
native, full-length proteins in cells. We show that the generated sABs are able to
pull-down endogenous proteins from mammalian cell extracts along with their natural
binding partners. We developed a method of utilizing our high affinity and specificity
binders as fluorescently labeled tools to visualize target proteins in their native
environment in the cells without the need of secondary antibodies or blocking reagents.
Our system also includes a method of efficient delivery of generated antibodies to
living cells, where they can perform their function. The sABs have been successfully
used for altering biological processes in a controllable manner.
PB-088
In vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds
to target receptors, proteases and trophic factors
Tai Kubo1, Mohammed Naimuddin1, Seigo Ono1
1National Institute of Advanced Industrial Science and Technology (AIST)
In vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds
to target receptors, proteases and trophic factors Small molecule natural products
are precious resources for drug discovery. During millions of years of evolution,
natural products must have been exposed to various selection pressures and have been
refined in structure and function to obtain the present features. In some peptide
neurotoxins, however, the basic molecular scaffold mainly configured by disulfide
(S-S) bridges and/or alpha/beta structures, is strictly conserved within each family
even under the evolution pressure. On the other hand the loop regions, which are not
heavily involved in scaffold formation, are highly diverged. This mode of molecular
evolution named ‘accelerated evolution’, is reasonable to quickly adapt to the vigorous
change of the environment. The evolutionally selected scaffold is compact harboring
both rigidity and flexibility in nature, and it may support a topology appropriate
for target recognition and selective interaction. Inspired by the system, we designed
random peptide libraries from the peptide neurotoxins of the accelerated evolution.
A three-finger (3F) shaped snake neurotoxin consists of huge family evolved by accelerated
gene evolution. We prepared a 3F-peptide library by introducing random sequences in
each fingertip. Another random peptide library with an ICK (inhibitor cystine knot)
motif was prepared based on a neurotoxin GTx1-15 from spider; originally identified
as a T-type Ca2+ channel modulator. Each library was subjected to in-vitro evolution
directed to specific target molecules. For the 3F-peptide library cDNA display method
was applied to select binders. When interleukin-6 (IL-6) receptors were targeted,
the selected 3F peptides showed binding affinities (Kd ∼100 nM) comparable to the
native ligand IL-6. When trypsin was targeted, peptides with serine protease inhibitor
activities similar to STI and BPTI (Ki ∼30 nM) were isolated. Specific binders to
a trophic factor VEGF were also generated from the 3F library. To target membrane
proteins, we developed a unique in-vitro evolution system, and named it as the PERISS
(intra periplasm secretion and selection) method. In the system, target membrane proteins
are expressed in inner membrane of E. coli and peptides are secreted to the periplasmic
space, in between the inner and outer membranes; and the space is served for interaction
and selection. The PERISS method enabled us to identify a peptide specific to muscarinic
receptor m2 subtype from the ICK peptide library. In conclusion, it was proved that
the library designed from the scaffold of peptide toxin, which evolved in the mode
of accelerated gene evolution, has pluripotency in target recognition, interaction
and even bioactivity. The library, when combined with in-vitro evolution technologies,
may open a new platform to develop antibody alternatives.
PB-089
Stereoselectivity of PAL under non-optimal conditions
Andrea Varga1, Botond Nagy1, Melinda Miklós1, Florin-Dan Irimie1, László Poppe1, Csaba
Paizs1
1Biocatalysis and Biotransformation Research Group, Department of Chimie, UBB
Phenylalanine ammonia lyase from Petroselinum cripsum (PcPAL) belongs to the class
of enzymes containing 4-methylideneimidazole-5-one (MIO) as a prostetic group and
it is responsible for the conversion of L-phenylalanine into trans-cinnamic acid.
This reaction is reversibile under high ammonia concentration. 1
We analyzed several factors that can influence the enantioselective synthesis of nitrophenylalanine
mediated by whole cells as well as purified MIO-containing and MIO-less PcPALs. First
we investigated the behaviour of the enzymes depending on the ammonia concentration.
We also inspected the influence of the pH on the PcPAL catalyzed biotransformations.
Based on our results, we concluded that variation of ammonia concentration and the
pH leads to decrease of enantioselectivity, suggesting that PcPAL is able to catalyze
the formation of both L- and D- enantiomers of electron-deficient structures.
Scheme 1.
PcPAL mediated amination of trans-nitro-cinnamate in non-stereoselective manner
Acknowledgements:
AV thanks for the financial support of the Sectoral Operational Programme for Human
Resources Development 2007-2013, co-financed by the European Social Fund, under the
project POSDRU/159/1.5/S/137750 and of the Romanian National Authority for Scientific
Research, CNCSUEFISCDI, project number PN-II-ID-PCE- 2011-3-0799. The authors also
thank the support to Jody L.
References:
1 M. M. Heberling, et al. Current Opinion in Chemical Biology 2013, 17:250–260
PC-001
Biochemical Characterization and Amino Acid Sequence Analysis of Thermostable Endo-ß-1,
4-glucanase from Trichoderma viride
Nidhee Chaudhary1, Monendra Grover2
1Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector-125, 2.-1Centre
for Agricultural Bioinformatics, IASRI
Abstract In the present investigation, Endo-β-1,4-glucanase has been purified to homogeneity
from Trichoderma viride using (NH4)2SO4 fractionation, DEAE-cellulose chromatography
and CM-cellulose chromatography. The purified enzyme had a molecular mass of 44.67
kDa with pH and temperature optima 4.8 and 500C, respectively, and thermostability
upto 500C for 48h. The purified enzyme showed a novel N-terminal 15 amino acid sequence
‘SYPNKQPYGPSGFWM’. However, T. viride endo- β-1,4-glucanase, like all microbial cellulase
appears to have a conserved ‘SG’ amino acid sequence at an identical position in the
N-terminal domain. The properties of the N-terminal 15 amino acid sequence were also
predicted computationally. This analysis showed that N-terminal sequence of the enzyme
is unstable. The N-terminal sequence also showed potential cleavage sites by different
proteases which may contribute to its instability. The secondary structure analysis
showed that the N-terminal sequence has 40% of the 15 a.a. sequence in extended strand
and 60% in random coil conformation. The N-terminal sequence was also analyzed for
potential phosphorylation sites. While no potential serine and threonine sites were
predicted, two tyrosine phosphorylation sites were predicted in the N-terminal sequence.
The N-terminal sequence was also examined for the presence of kinase specific phosphorylation
sites. The results showed the presence of one potential site which may be phosphorylated
by PKC at position 1 of the N-terminal sequence. The analysis for the prediction of
the presence of OGlcNAc sites revealed that two such sites may potentially be present
in the sequence. We have also predicted the ligand binding site in the N-terminal
sequence of the protein.
Keywords:
Endo-β-1,4-glucanase, Trichoderma viride, N-terminal amino acid sequence, phosphorylation,
secondary structure
PC-002
Modulation of the enzymatic activity of protein arginine methyltransferase 1 by small
molecules
Wey-Jinq Lin1
1National Yang-Ming University
Protein arginine methylation catalyzed by protein arginine methyltransferases (PRMTs),
is a pivotal protein post-translational modification involved in a growing number
of physiological and pathological processes including signal transduction, proliferation,
differentiation and malignancy. PRMT1 accounts for the majority of protein arginine
methyltransferase activity in mammalian cells and, in consistence, a large amount
of cellular substrates have been identified. Several studies have reported that the
activity of PRMT1 changes upon stimulation in various cellular processes. In mammalian
cells, PRMT1 exists in a high molecular weight complex. The interacting partners of
PRMT1, such as antiproliferative proteins BTG1 and BTG2, protein phosphatase 2A, the
orphan receptor TR3, and CCR4-associated factor 1(hCAF1) are shown to play a role
in modulating the methyltransferase activity and the substrate selectivity of PRMT1.
Due to the pivotal roles of PRMT1 in physiological and pathological conditions, intensive
efforts have been put on the search of small synthetic chemical molecules which can
efficiently modulate the activity of PRMT1 for the potential development of therapeutics.
In light of this, the intracellular small molecules that either transmit extracellular
stimulation or act as cofactor to dictate the activity of PRMTs in cells are still
poorly understood. Our study focused on examining how cellular ions might affect the
activity of PRMT1 and found that divalent and monovalent ions differentially modulated
the catalytic activity of PRMT1 toward different substrates.
PC-003
Oligomerisation properties of light-dependent protochlorophyllide oxidoreductase
Michal Gabruk1, Anna Piszczek1, Bozena Skupien-Rabian1, Sylwia Kedracka-Krok1,2, Jerzy
Kruk1, Beata Mysliwa-Kurdziel1
1Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 2Malopolska
Centre of Biotechnology, Jagiellonian University
Light dependent protochlorophyllide oxidoreductase (POR, E.C. 1.3.1.33) is a key enzyme
on chlorophyll biosynthesis pathway. It catalyses the conversion of protochlorophyllide
(Pchlide) to chlorophyllide using NADPH in a light-dependent manner. This special
property makes the enzyme unique and interesting subject of study. In our research,
we have obtained recombinant PORA from A. thaliana and a fusion protein of PORA-GFP.
We have confirmed that the enzyme works as an oligomer using different methodological
approaches. Using native gel electrophoresis, we have shown that the enzyme simultaneously
forms monomers, dimers and much larger oligomers without substrates in the lipid-free
environment. However, addition of Pchlide, but not NADPH, promotes formation of the
oligomers. In order to get insights into the structure of Pchlide:POR:NADPH oligomer,
we have applied cross-linking assay followed by mass-spectrometry analysis to identify
which domains of the enzyme interact with each other. We have analysed our results
with respect to the homology model of POR since there is no crystal structure of the
enzyme known. Taking advantage of the broad spectrum of applied techniques, we have
been able to propose the model of POR oligomerization.
Acknowledgement:
This work was supported by the grant Preludium 5 NCN2013/09/N/NZ1/00200 obtained form
the National Science Centre.
PC-004
Preparation of insect prothoracicotropic hormone with complicated disulfide-bond structure,
by the heterologous expression in Brevibacillus choshinensis
Kazuki Saito1, Tadafumi Konogami1, Yiwen Yang1, Yusuke Yamashita1, Masatoshi Iga1,
Tamari Hoshikawa1, Hiroshi Kataoka1
1Dept. of Integrated Biosciences, Grad. Sch. of Frontier Sciences, Univ. of Tokyo
Prothoracicotropic hormone (PTTH) is one of the most important neuropeptide regulators
for insect molting and metamorphosis. However, preparation of its recombinant protein
has hardly been successful, because it is a homodimer protein with very complicated
disulfide-bond structure. For example, silkworm PTTH has three intramolecular disulfide
bonds in its 109-residue polypeptide chain, and the two chains are further linked
by an additional intermolecular disulfide bond to form the homomeric dimer. Although
the recombinant silkworm PTTH was previously expressed in Escherichia coli, the product
was obtained only in precipitation fractions, and refolding of the precipitated protein
provided the active dimer PTTH in very poor yield. Under such reductive conditions
as in cytosol of the E. coli cells, formation of the correct disulfide-bond arrangement
must be difficult. Alternatively, for the heterologous expression of the silkworm
PTTH, we employed Brevibacillus choshinensis (formally referred to as Bacillus brevis),
which has achieved good results in expression of various disulfide-bond-containing
proteins. In this study, the silkworm PTTH was expressed in the Brevibacillus cells
with an additional His6-tag sequence at the C-terminus, for easier detection and purification.
First of all, since the Brevibacillus bacteria are equipped with a secretory system
of the expressed proteins, a secretory signal sequence to be attached before the silkworm
PTTH was carefully selected. Among four candidates in a commercially-available kit,
a signal sequence derived from an intrinsic cell-wall protein MWP gave better results
in expression levels of the protein. Second, incubation time of the cells was optimized,
because an oligomerization state of the secreted PTTH in the cell culture medium changed
with the time. In the medium, various PTTH oligomers including a monomer and a dimer
were initially observed, but higher oligomers became a major portion of the secreted
product after longer incubation than 48 h. Incubation for 24–36 h may be suitable
for obtaining the native dimer form of the silkworm PTTH. To remove the undesired
monomer and higher oligomers, which mostly retained free sulfhydryl groups, the secreted
proteins were treated with maleimide-PEG2-biotin. In the purification using a Ni+-NTA
column, the dimer of the His6-tagged silkworm PTTH was eluted with an imidazole gradient,
separately ahead of other biotinylated proteins, probably due to interaction of the
PEG2 spacer with the Ni+-NTA groups of the resin. After the reversed-phase HPLC purification,
the final product showed a single band on the non-reductive SDS-PAGE, and it had adequate
ecdysone-releasing activity from isolated silkworm prothoracic glands. The Brevibacillus
bacteria are most promising host cells for the heterologous production of the insect
PTTH.
PC-005
Role of the disulfide bridges in the transmembrane region of the insect prothoracicotropic-hormone
receptor, Torso
Tadafumi Konogami1, Yiwen Yang1, Mari H. Ogihara1, Juri Hikiba1, Hiroshi Kataoka1,
Kazuki Saito1
1Dept. of Integrated Biosciences, Grad. Sch. of Frontier Sciences, Univ. of Tokyo
Torso is an insect cellular-membrane protein, which was recently identified as a receptor
for prothoracicotropic hormone (PTTH). Although PTTH is one of the important regulatory
molecules in insect molting and metamorphosis, activation mechanism of Torso by the
ligand has not been elucidated yet. In this study, an oligomerization manner of the
silkworm Torso was examined, using heterologous expression in Drosophila S2 cultured
cells, because Torso is a single-polypeptide receptor tyrosine kinase (RTK), and activation
of such RTKs is often triggered by the ligand-induced receptor dimerization on the
cellular membrane. When activated with silkworm PTTH, dimerization of the silkworm
Torso in the S2 cells was observed, using a cross-linking reagent BS3, and the subsequent
receptor autophosphorylation and downstream ERK phosphorylation were also detected.
Surprisingly, however, the Torso dimerization was revealed to occur even without the
ligand stimulation, while the autophosphorylation and the ERK phosphorylation were
held in response to the stimulation. When fractionated by non-reductive SDS-PAGE,
the silkworm Torso showed an obvious dimer band, in addition to a faint monomer band,
both with and without the PTTH simulation, even though the receptor was not treated
with the cross-linking reagent. This indicates that the Torso protein is expressed
originally as a disulfide-bond-linked dimer. In addition, by examining oligomerization
states of several truncation and substitution mutants, cysteine residues in the transmembrane
region were found to participate in the intermolecular disulfide bridges, linking
the two receptor molecules in the dimer. When all of the three cysteines in the transmembrane
region were replaced by phenylalanines, the disulfide-bond-linked Torso dimerization
was not observed, but spontaneous, ligand-independent association of the Torso molecules
was detected using the cross-linker BS3. This spontaneous dimerization caused the
apparent Torso autophosphorylation, but it could not induce the downstream ERK phosphorylation.
Consequently, without the intermolecular disulfide bridges, Torso loses its responsiveness
to the PTTH stimulation. In conclusion, the disulfide bridges in the transmembrane
region may play a role to preserve suitable relative position between the two Torso
molecules, which could induce ligand-dependent autophosphorylation leading to activation
of the downstream signaling pathways in the cells.
PC-006
Structural study of the yeast enzyme neutral trehalase Nth1 and pNth1:Bmh1 protein
complex
Miroslava Kopecka1,2, Zdenek Kukacka3, Petr Man3, Tomas Obsil2, Veronika Obsilova2
12nd Faculty of Medicine, Charles University in Prague, 2Institute of Physiology of
the Czech Academy of Sciences, 3Institute of Microbiology of the Czech Academy of
Sciences
The yeast enzyme neutral trehalase (Nth1, EC 3.2.1.28) from Saccharomyces cerevisiae
hydrolyses the non-reducing disaccharide trehalose which serves as an energy source
and a universal stress protectant in many different organisms. Enzymatic activity
of Nth1 is enhanced by the yeast 14-3-3 protein (Bmh1 and Bmh2) binding in a phosphorylation-dependent
manner. Nth1 activity is also regulated by Ca2+ binding to the EF-hand-like motif
containing domain of Nth1 [1].The native TBE PAGE and analytical ultracentrifugation
show that Nth1 forms very stable complexes with Bmh1 and Bmh2 [1]. To study the structure
of Nth1 alone and its complex with the 14-3-3 protein we used circular dichroism,
H/D exchange coupled to mass spectrometry, chemical cross-linking [2] and small angle
X-ray scattering (SAXS) [3]. At the same time protein crystallography of Nth1 alone
and its complex with Bmh1 is performed.The low resolution structure of pNth1:Bmh1
protein complex revealed that binding of Bmh1 induces a rearrangement of the whole
Nth1 molecule and that the region containing the EF-hand motif forms a separate domain
which interacts with both Bmh1 and catalytic domain of Nth1. We proved that integrity
of the EF-hand motif is crucial for the Bmh1 mediated activation of Nth1 and Ca2+
binding. Our data suggest that the EF hand-like motif functions as the intermediary
through which Bmh1 modulates the function of the catalytic domain of Nth1. These structural
changes probably enable the substrate entry into the enzyme active site [3]. Our study
of 14-3-3 protein complex with the fully active enzyme Nth1 offers a unique structural
view of Nth1 activation enabling us to better understand the role of the 14-3-3 proteins
in regulation of other enzymes.
This work was supported by the Czech Science Foundation (Project P207/11/0455) and
by Grant Agency of Charles University (Grant 644313).
1. D. Veisova, E. Macakova, L. Rezabkova, M. Sulc, P. Vacha, H. Sychrova, T. Obsil,
V. Obsilova, Biochem. J. 443, (2012), 663 – 670.
2. E. Macakova, M. Kopecka, Z. Kukacka, D. Veisova, P. Novak, P. Man, T. Obsil, V.
Obsilova, Biochim. Biophys. Acta. 1830, (2013), 4491 – 4499.
3. M. Kopecka, D. Kosek, Z. Kukacka, L. Rezabkova, P. Man, P. Novak, T. Obsil, V.
Obsilova, J. Biol. Chem. 289, (2014), 13948 – 13961.
PC-007
Development and use of a molecular purge valve to maintain reduction/oxidation balance
in synthetic biochemistry systems
Tyler Korman1, Paul Opgenorth1, James Bowie1
1Department of Chemistry and Biochemistry, University of California Los Angeles
The assembly of self-regulating synthetic biochemical pathways in vitro has great
potential as alternative catalysts for the high-yield production of low value/high
volume commodity chemicals from biomass. High yields of low-value/high volume compounds
that are required for economic viability is particularly difficult via traditional
in vivo metabolic engineering of microbes due to competing biochemical pathways and
toxicity. We have developed an alternative approach, called synthetic biochemistry,
where the glycolysis pathway of central metabolism is reconstituted in vitro with
an anabolic pathway that can produce useful compounds at high yield. In the specific
synthetic biochemistry system described, reducing equivalents, ATP, and carbon from
glycolysis are funneled through the anabolic mevalonate pathway to produce the monoterpene
limonene from glucose. The successful implementation of the in vitro pathway required
development of a molecular purge-valve consisting of an NAD+ and NADP+ specific reductase
(ie wild-type and mutant pyruvate dehydrogenase), and NADH oxidase, NoxE, to maintain
proper NADP+/NADPH cofactor balance while allowing continuous carbon flux. We find
that the purge-valve concept is readily transportable to other NAD(P)H generating
steps in central metabolism and can be used to convert glucose to limonene at high
yield.
PC-008
Evolution of Structure and Mechanistic Divergence in Di-Domain Methyltransferases
from Nematode Phosphocholine Biosynthesis
Soon Goo Lee1, Joseph Jez1,
1Washington University in St. Louis
The phosphobase methylation pathway is the major route for supplying phosphocholine
to phospholipid biosynthesis in plants, nematodes, and Plasmodium. In this pathway,
phosphoethanolamine N-methyltransferases (PMT) catalyzes the sequential methylation
of phosphoethanolamine to phosphocholine. In the PMT, one domain (MT1) catalyzes methylation
of phosphoethanolamine to phosphomonomethylethanolamine and a second domain (MT2)
completes the synthesis of phosphocholine. The x-ray crystal structures of the di-domain
PMT from the parasitic nematode Haemonchus contortus (HcPMT1 and HcPMT2) reveal that
the catalytic domains of these proteins are structurally distinct and allow for selective
methylation of phosphobase substrates using different active site architectures. These
structures also reveal changes leading to loss of function in the vestigial domains
of the nematode PMT. Divergence of function in the two nematode PMT provides two distinct
anti-parasitic inhibitor targets within the same essential metabolic pathway. The
PMT from nematodes, plants, and Plasmodium also highlight adaptable metabolic modularity
in evolutionarily diverse organisms.
PC-009
Glycoside hydrolase family18 chitinase from the stomach of fish: characteristics of
isozymes
Masahiro Matsumiya1, Hiromi Kakizaki1, Mana Ikeda1
1College of Bioresource Sciences, Nihon University
Chitinases (EC 3.2.1.14) are enzymes that randomly hydrolyze β-1,4 glycosidic bonds
of chitin and produce N-acetylchitooligosaccharide ((GlcNAc)n) that has various physiological
functions such as immunostimulatory activity. Most of fish takes crustacean such as
shrimp and crab as food. Therefore, the fish has chitinase in the stomach to chemically
disrupt the chitinous envelope of crustacean. Four chitinase isozymes (42-60 kDa),
PaChiA[1] and PaChiB[2], and PtChiA and PtChiB, [3] were purified from the stomach
of silver croaker Pennahia argentatus and threeline grunt Parapristipoma trilineatum,
by ammonium sulfate fractionation and column chromatographies, respectively. All the
chitinases were stable and showed activity in the acidic pH range (pH3-5). PaChiA
and PtChiA preferentially degraded the second glycosidic bond from the non-reducing
end of (GlcNAc)n and PaChiB and PtChiB had a preference for the third glycosidic bond
of those. All the chitinases showed different substrate specificity toward insoluble
long substrates. Moreover, chitinase cDNAs (PaChi-1 and PaChi-2) encoding PaChiA and
PaChiB, and cDNAs (PtChi-1 and PtChi-2) encoding PtChiA and PtChiB were obtained by
cDNA cloning using the RT-PCR and RACE method. The deduced amino acid sequences of
all the chitinase cDNAs contained N-terminal signal peptide, GH family 18 catalytic
domain, linker region, and chitin-binding domain. Phylogenetic tree analysis of vertebrate
chitinase revealed that fish stomach chitinases form unique chitinase isozyme groups,
acidic fish chitinase-1 (AFCase-1) including PaChiA and PtChiA, and acidic fish chitinase-2
(AFCase-2) including PaChiB and PtChiB, which was different from an acidic mammalian
chitinase (AMCase) group.[3,4] The previously reported purified fish stomach chitinases[5]
can also be classified into two chitinase isozyme groups, AFCase-1 and AFCase-2, by
the N-terminal amino acid sequence. This study suggested that fish have excellent
chitin degrading enzymatic system in which two different chitinases isozyme groups,
AFCase-1 and AFCase-2, with different degradation patterns are expressed in the stomach.
[1] M. Ikeda, K. Miyauchi, A. Mochizuki, and M. Matsumiya, Purification and characterization
of chitinase from the stomach of silver croaker Pennahia argentatus. Protein Expr.
Purif. 65, 214–222 (2009).
[2] M. Ikeda, K. Miyauchi, and M. Matsumiya, Purification and characterization of
a 56 kDa chitinase isozyme (PaChiB) from the stomach of silver croaker Pennahia argentatus.
Biosci. Biotechnol. Biochem. 76, 971–979 (2012).
[3] M. Ikeda, Y. Kondo, and M. Matsumiya, Purification, characterization, and molecular
cloning of chitinases from the stomach of the threeline grunt Parapristipoma trilineatum.
Process Biochem. 48, 1324–1334, (2013).
[4] M. Ikeda, D. Shirase, T. Sato, M. Ueda, S. Hirabayashi, and M. Matsumiya, Primary
structure and enzymatic properties of chitinase isozymes purified from the stomach
of the marbled rockfish Sebastiscus marmoratus. J. Chitin Chitosan Sci.2, 106-116
(2014).
[5] M. Matsumiya, Y. Arakane, A. Haga, S. Muthukrishnan, and K. J. Kramer, Substrate
specificity of chitinases from two species of fish, greenling, Hexagrammos otakii,
and common mackerel, Scomber japonicus, and the insect, tobacco hornworm, Manduca
sexta, Biosci. Biotechnol. Biochem., 70, 971-979 (2006).
PC-010
A bifunctional cold active lipase with protease activity isolated from an Antarctic
yeast, Glaciozyma antarctica PI12
Mohd Shukuri Mohamad Ali1,2, Ira Maya Haris1,2, Raja Noor Zaliha Raja Abd Rahman1,2,
Mahiran Basri3, Abu Bakar Salleh1,2
1Enzyme and Microbial Technology Research Center, 2Faculty of Biotechnology and Biomolecular
Sciences, 3Faculty of Science
Recently, the enzymes produced by psychrophilic organisms have gained huge interest
especially in the studies of temperature adaptation of the protein. Previously, a
cold-adapted yeast, Glaciozyma antarctica PI12 was isolated from a marine environment
in Antarctica and the yeast was known to produce lipolytic and proteolytic enzymes.
A gene encoding a unique recombinant bifunctional enzyme (LipPI12) with cold active
lipase with protease activity was successfully expressed, purified and characterized.
Temperature profile of the bifunctional LipPI12 enzyme showed that the lipase functions
optimally at 20°C whereas the protease was more active at 40°C. pH profile showed
that both LipPI12 lipase and protease were active at near neutral condition. Activity
of LipPI12 lipase and protease were also activated in the presence of CaCl2 but its
protease counterpart seemed to be more active in the presence of ZnCl2. Effect of
surfactants showed LipPI12 lipase was activated by Tween 80 and SLS and in contrast,
LipPI12 protease was almost deactivated in all surfactants tested. The presence of
organic solvents did not affect both the lipase and protease activities. The lipase
was more stable at solvents with higher log P value whereas the protease was slightly
activated at low log P value particularly with dimethylsulfonyl. Inhibitor studies
revealed that LipPI12 lipase was partially inhibited with EDTA and PMSF whereby the
LipPI12 protease was inhibited by pepstatin, EDTA and PMSF. LipPI12 enzyme was successfully
crystallized via vapour diffusion method. Crystal of LipPI12 enzyme was diffracted
via synchrotron radiation. The three-dimensional structure of cold-adapted PI12 provided
insight into cold adaptation and better understanding of the structural properties
of LipPI12 enzyme. The bifunctional properties of the enzyme could be potential candidate
for low temperature industrial application.
PC-011
Conformation-specific antibodies as enhancers and inhibitors of phosphatase activity
of DEP 1
Malgorzata Nocula-Lugowska1, Mateusz Lugowski1, Anthony A. Kossiakoff1
1The University of Chicago
DEP-1 (CD148/PTP-η) is a transmembrane receptor-like protein tyrosine phosphatase
(PTP) that has been implicated in the density-dependent regulation of cell growth,
differentiation and transformation. It counteracts protein kinases by dephosphorylating
a number of their substrates as well as the kinases themselves, thus potentially controlling
the specificity of signals. For example EGFR, VEGFR 2, Met, PDGF β receptor have been
shown to be dephosphorylated by this phosphatase. DEP-1 has been shown to act as a
tumor suppressor and it has been proposed as a molecular target in anti-angiogenesis
therapy. As a result, both enhancers and inhibitors of DEP-1 activity have the potential
of elucidating pathways responsible for abnormal cell behavior. We generated synthetic
antibodies against intracellular catalytic domain of DEP-1 that act as modulators
of the enzyme’s phosphatase activity. By applying a combination of selection pressures
an array of antibodies has been raised from phage display libraries of Fab fragments
which are capable of either enhancing or inhibiting DEP-1 activity. In phosphatase
assays with catalytic domain of DEP-1 the antibodies demonstrate non-competitive or
mixed kinetics. The crystal structure of DEP-1-inhibitor complex shows that this antibody
binds to the part of the protein that is distant from the active site and acts by
locking the enzyme in the non-natural catalytically inactive state by hindering the
closure of the WPD loop which is crucial for the reaction to occur. By contrast, as
judged from the crystal structure of a complex of DEP-1 with the antibody that enhances
its phosphatase activity, this antibody seems to act by stabilizing the naturally
found active state of DEP-1 with WPD loop in the closed conformation. The antibodies
are also able to recognize DEP-1 in cells, as they stain DEP-1 in immunofluorescence
experiments. To test the applicability of raised antibodies in cells the activator
was additionally used to pull down full-length endogenous DEP-1 after being delivered
to live cells. Inhibition and enhancement of DEP-1 activity by locking the enzyme
in conformations which are either natural or imposed by allosteric binding of antibodies
seems to be a mechanism that can be utilized to modulate activity of other tyrosine
phosphatases.
PC-012
Investigating Acinetobacter baumannii pathogenesis: crystal structure of WbjB epimerase
from a polysaccharide biosynthesis cluster
Bhumika S. Shah1, Karl A. Hassan A. Hassan1, Heather E. Clift1, Stephen J. Harrop2,
Ian T. Paulsen1, Bridget C. Mabbutt1
1Department of Chemistry and Biomolecular Sciences, Macquarie University, 2School
of Physics, University of New South Wales
Acinetobacter baumannii is a multi-drug resistant opportunistic pathogen emerging
as a major health threat in clinical and community settings worldwide. Recent comparative
genome analysis across Acinetobacter spp. highlights that large portions of its genetic
material are not fixed, but rather held within a mobile pangenome. Acquired through
lateral transfer, these mobilized genes cluster within genomic islands (GIs), encoding
highly adaptive traits such as virulence, pathogenicity and drug resistance. I have
analyzed three new A. baumannii genome sequences derived from Australian hospitals
and indigenous sources, so complementing previously studied genomes from USA and Europe.
My work identified GIs resident within these strains, clusters of novelty account
for 9-16% of each genome. One GI of interest, found in an A. baumannii strain from
a community-acquired infection in an indigenous settlement in Northern Australia,
clearly encodes discrete steps of lipopolysaccharide (LPS) biosynthesis, so likely
influencing virulence factors for its host pathogen. I have focused on the encoded
epimerase WbjB, a member of the extended shortchain dehydrogenase/reductase family
(SDR). The crystal structure solved to 2.65 Å reveals an unusual hexameric form of
SDR, with cofactor NADP bound. Assay of WbjB indicates responsiveness to UDP sugar
molecules such as UDP-GlcNAc and UDP-Glc. I describe screening and first knock out
strains created in A. baumannii. These aim to assess the role of the GI cluster in
carbon metabolism, as well as its impact on biofilm formation. Phenotypic differences
of wild type and knock out strains will subsequently be investigated to identify adaptive
traits attributable to this LPS cluster.
PC-013
Role of the Hydrogen Bonding Interactions in the O2 Sensitivity of HIF-Prolyl Hydroxylase
(PHD2)
Serap Pektas1, 2, Michael Knapp1
1University of Massachusetts Amherst, 2Recep Tayyip Erdogan University
Oxygen homeostasis is regulated by hypoxia inducible factor, a transcription factor.
When the oxygen level becomes too low (hypoxia), hypoxia-inducible-factor 1 (HIF-1α)
activates the expression of over a hundred genes, associated with angiogenesis, erythropoiesis,
VEGF (vascular endothelial growth factor), cell migration, and energy metabolism etc.
HIF-1α cellular level is highly dependent on oxygen concentration and regulated by
oxygen sensor enzyme, HIF prolyl hydroxylase (PHD2). PHD2 is a 2-oxoglutarate, non-heme
Fe2+ dependent dioxygenase, which require O2 for catalytic activity. PHD2 regulates
the HIF-1α cellular level by hydroxylating two proline residues in ODD domain of HIF-1α,
which targets HIF-1α for proteasomal degradation. In order to understand how O2 activation
of PHD2 enzyme works, we investigated the effect of residues, which have hydrogen
bonding interactions with O2 ligand of PHD2. Point mutations and steady state kinetic
assays were performed and compared with wild type PHD2. Eliminating certain hydrogen
bonding interactions of O2 ligand led increased turnover rate at low O2 concentration.
Our results revealed that hydrogen bonding interactions with O2 ligand determines
the O2 sensitivity of PHD2.
PC-014
New pharmacological therapies against congenital erythropoietic porphyria (CEP)
Pedro David Urquiza1, Ana Laín1, Arantza Sanz1, Juan Manuel Falcón1, 2, Oscar Millet1
1CIC bioGUNE, 2Ikerbasque
Congenital erythropoietic porphyria (CEP) is produced by deleterious mutations in
uroS gene. Among the most aggressive mutations, C73R drastically reduces the activity
and stability of uroporphyrinogen III synthase enzyme (UROIIIS), present in almost
one of third of all the reported CEP cases. Previous studies in our laboratory demonstrated
that the catalytic activity UROIIIS was fully restored, by incorporating residues
prone to interact with 73R to stabilize the hinge region, as well as a modulated increase
in the kinetic stability of the enzyme. These results provide an unprecedented rationale
for a destabilizing missense mutation. At the present time we are screening different
molecular chaperones (library of 2500 compounds) as a therapy against CEP which should
be able to upregulate the protein’s homeostasis by binding to the enzyme in order
to stabilize the folded conformation. At the moment, some chaperones stabilize the
hotspot C73R in vitro, the results were monitored by circular dichroism (CD) and nuclear
magnetic resonance (NMR). On the other hand, we are studying the effects of the compounds
in UROIIIS-GFP mutant cells. The results obtained at HC automated fluorescent microscope
suggest that the compounds enter into the cells and interact with the protein UROIIIS-C73R-GFP.
This could indicate that they could be acting as chaperones.
PC-015
Delicate Balance of Noncovalent Forces Control the Electron Transfer Complex between
Ferredoxin and Sulfite Reductase to Optimize Enzymatic Activity
Juyaen Kim1, Misaki Kinoshita1, Takahisa Ikegami1,2, Genji Kurisu1, Yuji Goto1, Toshiharu
Hase1, Young-Ho Lee1
1Institute for Protein Research, Osaka University, 2Yokohama City University
Plant sulphite reductase (SiR) forms an electron transfer complex with ferredoxin
(Fd) for the reductive conversion of sulphite to sulphide. Although previous studies
have highlighted electrostatic interactions between oppositely-charged residues of
the two proteins, detailed thermoenergetics of the intermolecular interaction for
the complexation remains unknown. We herein carried out isothermal calorimetry of
Fd:SiR complex formation at various NaCl concentrations. Driving force plot constructed
from calorimetry showed that the complex was thermodynamically stabilized by both
enthalpy and entropy through favourable electrostatic and non-electrostatic interactions.
Increasing NaCl concentrations weakened interprotein affinity and contribution of
the negative enthalpy changes became decreased, while no such significant decrease
was found in the contribution of positive entropy changes. Furthermore, a negative
heat capacity change obtained from the enthalpy changes at distinct temperature indicated
a contribution of hydrophobic interactions. These findings suggested that both electrostatic
and non-electrostatic interprotein interactions were energetically important for the
complex formation. Fd-dependent SiR activity assay revealed a bell shaped activity
curve with a maximum under a certain NaCl concentration, while the methyl viologen-dependent
assay of SiR exhibited a profile of saturating curve, suggesting that an optimized
interprotein interaction is a crucial factor in control of Fd-dependent-SiR activity.
A residue-based NMR measurement of 15N-labeled Fd upon complex formation with SiR
revealed that charged and non-charged residues were differentially contributed in
the complex formation depending on NaCl concentrations. We proposed that non-electrostatic
forces were also critical for forming the Fd:SiR complex, and an optimized complex
conformation for maximum enzymatic activity was achievable by a delicate balance among
non-covalent intermolecular forces. These results may be extended for understanding
of complexation between redox proteins containing biased charge clusters.
PC-016
Ornithine transcarbamylase has a spatially extended active site as computationally
predicted
Lisa Ngu1, Kevin Ramos1, Nicholas DeLateur1, Penny Beuning1, Mary Jo Ondrechen1
1Department of Chemistry & Chemical Biology, Northeastern University
Understanding how an enzyme catalyzes a reaction is a fundamental problem in protein
science. Biochemical experimentation has revealed catalytic mechanisms of many enzymes;
however these studies have focused almost exclusively on amino acid residues in direct
contact with the reacting substrate molecule(s). Here we report on the computational
prediction and experimental verification of the importance of distal residues in enzyme
catalysis, using E. coli ornithine transcarbamylase as an example. Partial Order Optimum
Likelihood (POOL), developed at Northeastern University, is a machine learning technique
that only requires the tertiary structure of a protein to predict important catalytic
residues, based on computed, residue-specific electrostatic and chemical properties.
POOL has been shown to predict accurately the catalytic residues and to discern between
compact and spatially extended active sites. Dynamic conformational changes during
catalysis and strong electrostatic interactions give rise to significant coupling
between remote residues and the canonical active site residues of an enzyme. This
suggests that at least some enzyme active sites are spatially extended, with second-
and third- shell residues playing significant roles in catalysis. In this project,
we focus on ornithine transcarbamylase (OTC), for which dynamic processes are believed
to play a role in its catalytic mechanism. OTC is reported to undergo induced-fit
conformational changes upon binding carbamoyl phosphate, which affects the subsequent
binding of ornithine. Residues predicted by POOL to be catalytically important include
five in direct contact with the substrate, R106, H133, D231, C273 and R319. POOL also
predicted remote residues to form a spatially extended, triple-layer active site.
Guided by computational predictions and using site-directed mutagenesis and kinetics
assays of Asp140, His272, Glu299 and Arg57 variants, we show that these POOL-predicted
remote residues, located in the second and third layers, are important for catalysis.
Kinetics assays of wild-type OTC resulted in catalytic efficiencies of 170 ± 52 x
105 M-1s-1 for ornithine and 590 ± 86 x 105 M-1s-1 for carbamoyl phosphate, consistent
with previous studies. OTC variants R57A, D140N, Y160S, H272L and E299Q show significant
loss of catalytic efficiency. The results indicate that the charge on Glu299 and polarity
of His272 play roles in catalysis and demonstrate the importance of remote residues,
up to 10 Å away from canonical active site residues, for OTC activity. In addition,
we verify the power of POOL to predict important catalytic residues accurately. This
example, along with others, illustrates the importance of distal residues in natural
enzymes and also in protein engineering. Understanding how distal residues contribute
to catalysis has important implication for protein design for applications as diverse
as therapeutics, renewable energy systems, and green industrial chemical processes.
Supported by NSF MCB-1158176.
PC-018
Identification, Characterization, and Modification of Fatty Acid Alkyl Esterases Found
in Staphylococcus aureus
Benjamin Saylor1
1San Diego State University
Alternative energy is a major focus of current research efforts. Biodiesel, a mixture
of fatty acid alkyl esters, is one of the most versatile alternative fuels currently
in use. This is due to the fact that it is similar to gasoline and compatible with
diesel engines found throughout the existing global infrastructure. Biodiesel precursor
lipids are abundant in cultivated feedstock organisms such as algae and bacteria.
However, the standard process for converting oil to biodiesel is heat-intensive and
requires complete removal of water, reducing the overall net energy gained in its
production. Our work constitutes an attempt to explore enzymatic synthesis of biodiesel
from lipids such as those derived from emerging fuel crops. Previous literature describes
fatty acid alkyl ester formation in human patients with MRSA Staphylococcus aureus
wound lesions. These esters are formed by partially characterized esterase activity
from an unidentified source. We have identified two MRSA enzymes responsible for this
activity by using a combination of size exclusion chromatography, gas chromatography-mass
spectrometry, and mass spectrometric protein sequencing. These two highly similar
enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis
of fatty acid alkyl esters in aqueous conditions at or near room temperature. We have
demonstrated that other non-Staphylococcal lipases do not exhibit this behavior. We
have expressed these Staphylococcal esterases in E. coli, and shown via gas chromatography
that the expressed proteins catalyze the formation of fatty acid alkyl esters. Based
on sequence similarity to homologous proteins that have already been crystallized,
we have predicted a structure for these enzymes and have engineered mutant fusions
with higher rates of catalysis. Our design hypothesis is that increased avidity for
substrate molecules will yield a higher substrate concentration in the vicinity to
the enzyme. To increase substrate concentration we have designed and expressed one
of the enzymes as a chimeric fusion with the Drosophila melanogaster alcohol-binding
protein LUSH. GC-MS determination of biodiesel production rate indicates that the
chimeric fusion has a lower-order rate constant with respect to ethanol. In other
words, the fusion enzyme is less dependent on substrate concentration and is a superior
catalyst at low ethanol concentrations. This result indicates that the rationally
designed modification of binding avidity constitutes a potential avenue for improving
the ability of enzymes to catalyze reactions with low-concentration or low-solubility
substrates.
PC-019
Functional elements of a human antizyme essential for binding and inhibiting human
ornithine decarboxylase
Ju-Yi Hsieh1, Yi-Liang Liu1, Guang-Yaw Liu2, Hui-Chih Hung1
1Department of Life Sciences and Institute of Bioinformatics, National Chung Hsin,
2Institute of Microbiology & Immunology, Chung Shan Medical University, and Divis
Ornithine decarboxylase (ODC) plays an essential role in various biological functions,
including cell proliferation, differentiation and cell death. In this study, we revealed
that overexpression of ODC in HeLa and MCF-7 cells decreased cellular ROS (Reactive
oxygen species) after low dose of ultraviolet B radiation (UVB), leading autophagy
inhibited, and it was restored by knocking down ODC (shODC) in ODC overexpressing
HeLa and MCF-7 cells. Furthermore, the results demonstrated that AMPK was increased
after high dose of UVB radiation in ODC ovexpressing HeLa and MCF-7 cells, leading
autophagy induced and apoptosis inhibited. We demonstrated that knocked down autophagy
by shRNA (shAtg5, shBECN1, and shAtg12) and chloroquine (CQ) could enhance high dose
of UVB induced cell death in ODC overexpressing HeLa and MCF-7 cells. Here, we also
observed that knocked down ODC in ODC overexpressing HeLa and MCF-7 cells inhibited
autophagy and enhanced high dose of UVB radiation. Because of Atg12 can regulate cell
apoptosis and autophagy. Site directed mutagenesis was used to mutant the amino acid
which can regulate cell apoptosis and autophagy on Atg12, respectively in these two
ODC overexpressing cells. According to the results, mutated the amino acid which can
regulate apoptosis on Atg12 leading the cells more survival. Relatively, mutated the
amino acid which can regulate autophagy on Atg12 leading the cells died. Therefore,
inhibition of ODC and autophagy could be a promising strategy for adjuvant chemotherapy
in human breast and cervical cancers.
PC-020
Structure-Function Relationships of human Aldo-Keto Reductase 1B15, AN enzyme active
with 9-cis-Retinaldehyde
Joan Giménez Dejoz1, Michal H. H. Kolář2,3, Francesc Xavier Ruiz4, Isidro Crespo1,
Alexandra Cousido-Siah4, Alberto Podjarny4, Jindřich Fanfrlík2, Xavier Parés1, Jaume
Farrés1, Sergio Porté1
1Universitat Autònoma de Barcelona, 2Institute of Organic Chemistry and Biochemistry,
3Institute of Neuroscience and Medicine and Institute for Advanced Simulation, 4Institut
de Génétique et de Biologie Moléculaire et Cellulaire
Human aldo-keto reductase 1B15 (AKR1B15) is a recently identified member of the human
AKR family (Weber et al. J. Biol. Chem. 290, 6531-45, 2015). The enzyme displays 92%
sequence identity with AKR1B10, which efficiently catalyzes the reduction of a wide
variety of endogenous and exogenous carbonyls, such all-trans-retinaldehyde, and it
is linked to the development of several cancer types. In contrast, the enzymatic activity
and physiological role of AKR1B15 are still poorly understood. In this work, we have
improved the expression and purification of AKR1B15 using a detergent-free system.
AKR1B15 enzymatic activity has been characterized together with inhibitor screening
and structural modeling analyses. The results show that AKR1B15 is active towards
a variety of carbonyl substrates, including ketones and dicarbonyl compounds, with
lower Km and kcat values than those of AKR1B10. Moreover, AKR1B15 exhibits superior
catalytic efficiency towards 9-cis-retinaldehyde, the best substrate found for this
enzyme. Several typical AKR inhibitors do not significantly affect AKR1B15 activity.
Structural modeling reveals that AKR1B15 active site is smaller and more rigid than
the AKR1B10 pocket, mainly due to hydrophobic residue substitutions clustered in loops
A and C.
PC-021
Significance of protein substrate structure and dynamics in proteolysis: insights
from Kunitz-BPTI family canonical serine protease inhibitors
Olumide Kayode1, 2, Thomas R. Caulfield3, Ruiying Wang2, Devon Pendlebury2, Alexei
Soares4, Evette S. Radisky2
1Mayo Graduate School, 2Department of Cancer Biology, Mayo Clinic Cancer Center, 3Department
of Neuroscience, Mayo Clinic College of Medicine, 4Biology Department, Brookhaven
National Laboratory
Proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within
protein substrates; they have served as key model enzymes for studying the molecular
basis for catalytic power and specificity. Protease substrate specificity is most
often defined in terms of linear sequence motifs that flank the cleavage site; however,
the natural substrates of proteases are proteins with 3-dimensional shapes and complex
conformational dynamics that are not well represented by 1-dimensional sequence alone.
These structural and dynamical properties can impact recognition and binding of substrates
by proteases, as well as the efficiency of catalysis itself. In this study, we explore
the importance of substrate structure and dynamics for proteolysis using as our model
the cleavage of the Kunitz-BPTI family of canonical serine protease inhibitors by
mesotrypsin. Bovine pancreatic trypsin inhibitor (BPTI), an archetypal serine protease
inhibitor of the Kunitz family, has a high affinity interaction with trypsin, yet
its peptide bond hydrolysis is many orders of magnitude slower than other peptide
substrates. Mesotrypsin, a trypsin variant, has been shown to hydrolyze Kunitz family
inhibitors at accelerated rates; this is especially true of human Kunitz domain inhibitors.
Amyloid precursor protein inhibitor (APPI) and amyloid precursor like protein-2 (APLP2),
two human Kunitz domain family members, are hydrolyzed by mesotrypsin several hundred
times faster than BPTI. Here, we present a new, unpublished crystal structure of a
cleavage intermediate APLP2 bound to mesotrypsin, refined to 1.4Å resolution, revealing
a dramatic substrate conformational change we hypothesize to be required during cleavage
of a Kunitz domain. Using this structure along with published structures of APPI and
BPTI complexes, we have modeled acyl-enzyme intermediates of mesotrypsin, and we have
carried out molecular dynamic simulations that explore the transition of the initially
formed native-like acyl-enzyme through the conformational transformation that allows
the progression of the hydrolysis reaction. We further identify a specific hydrogen
bond, present in BPTI but not APPI, which forms a stabilizing feature of the BPTI
scaffold. Using site directed mutagenesis, we probe the contribution of this bond
to the proteolytic stability of BPTI. Collectively our data for these highly structured
substrates show that proteolysis rates are limited by a necessary conformational change
in the substrate as the reaction progresses. Rigid substrates possessing stabilizing
features that render them highly resistant to this conformational change are proteolyzed
more slowly than more flexible substrates of similar structure.
PC-022
Determinants for regioselectivity in Lytic Polysaccharide MonoOxygenases (LPMOs)
Barbara Danneels1, Magali Tanghe1, Henk-Jan Joosten2, Tom Desmet1
1Centre for Industrial Biotechnology and Biocatalysis, University of Ghent, 2Bioprodict
INTRODUCTION With the discovery of the lytic polysaccharide monooxygenases (LPMOs),
new light has been shed on the degradation of lignocellulosic biomass. LPMOs are copper
metalloenzymes that carry out the oxidative cleavage of the β-1,4-glycosidic bond,
generating new chain ends that can subsequently be processed by cellulases, boosting
the cellulose degradation. LPMOs have a β-sandwich conformation with a flat binding
surface, allowing for the enzyme to bind to crystalline cellulose. The Cu2+ ion, required
for activity, is located in a so-called “histidine brace”, in which the N-terminal
histidine is highly conserved. REGIOSELECTIVITY According to the carbon atom being
oxidized, 3 LPMO types are identified: type 1 and type 2 oxidizing at the C1 and the
C4 respectively, type 3 LPMOs oxidizing both the C1 and the C4 adjacent to the glycosidic
linkage. We were able to express a type-1 LPMO (Phanerochaete chrysosporium GH61D)
and a type-3 LPMO (Trichoderma reesei Cel61A) in P. pastoris. This has proven to be
very challenging, as LPMO activity requires a perfect cleavage of the signal sequence.
After activity assays on PASC, characteristic HPAEC-PAD traces were obtained which
will serve as a reference for engineering experiments. ENZYME ENGINEERING Using the
3DM database, a structure based multiple sequence alignment tool, it is possible to
identify residues specifically conserved in subsets of protein sequences. By defining
a subset for each LPMO type, we were able to identify residues contributing to regioselectivity.
These positions are now being rationally engineered in subsequent rounds of mutagenesis,
using TrCel61A as a template. The effect of the mutations will be determined by analyzing
the HPAEC-PAD trace released from PASC. The main goal is to investigate the possibility
of deleting the C4 specificity in a type 3 LPMO.
PC-023
Folding topology determines substrate binding order in the ribokinase superfamily
Alejandra Herrera-Morandé1, Victor Castro-Fernández1, Felipe Merino1, César Ramírez-Sarmiento1,
Francisco Fernández2, Cristina Vega2, Victoria Guixé1
1Departamento de Biología, Facultad de Ciencias, Universidad de Chile., 2Centro de
Investigaciones Biológicas (CIB-CSIC)
Folding topology determines substrate binding order in the ribokinase superfamily
1Alejandra Herrera-Morandé, 1Victor Castro-Fernández, 1Felipe Merino, 1César. A. Ramírez-Sarmiento,
2Francisco Fernández, 2Cristina Vega and 1Victoria Guixé. 1Departamento de Biología,
Facultad de Ciencias, Universidad de Chile, Santiago, Chile. 2Centro de Investigaciones
Biológicas (CIB-CSIC), Madrid, España Ribokinase superfamily comprises three enzyme
families: the ADP-dependent sugar kinases family, the ATP-dependent coenzyme kinases
family and the ATP-dependent sugar kinases family. In all these families there is
a large domain composed by a Rossmann motif but only the ATP-dependent enzymes have
a β-meander motif in the C-terminal end. Interestingly, these enzymes display an ordered
kinetic mechanism where the substrate that will be phosphorylated binds first to the
enzyme. The ADP-dependent enzymes present a topological re-ordering of the secondary
structural elements which produces an equivalent tertiary structure, which can be
thought as a non-circular permutation (NCP) of the β-meander region. These enzymes
also display an ordered kinetic mechanism but with an inversed order being the nucleotide
the first substrate to bind to the enzyme. As this β-meander region of the proteins
constitutes almost entirely the nucleotide binding site, and given that the permutation
is the major structural difference between ADP and ATP-dependent kinases, it could
the responsible for the nucleotide specificity. To test this hypothesis we introduce,
by permutation, an ATP-dependent topology in the homologous ADP-dependent glucokinase
from T. litoralis (perGK). Size exclusion chromatography and circular dichroism spectra
show that both the wild type and the permutated enzyme eluted as monomers with similar
hydrodynamic behavior, and have the same secondary structure content. Kinetic assays
employing ATP or ADP as substrate demonstrate that even in the presence of 10 mM ATP,
the perGK enzyme is not able to carry out the phosphoryl transfer. To test if the
NCP has an impact in the kinetic constants and substrate binding order we determine
the kinetic mechanism through classical protocols, involving initial velocity studies,
product inhibition and dead end inhibitors. The results demonstrate that the perGK
enzyme presents an altered substrate binding order compared to the wild type enzyme,
where glucose was the first substrate to bind to the enzyme and glucose-6-P the last
product to be released. Also, ligand-induced conformational changes were determined
in the crystal structures. The apo, the enzyme-glucose and enzyme-glucose-ADPβS structures
were determined at 2.14 Å, 1.95 Å and 2.44 Å resolutions, respectively. Structure
analysis reveals that glucose binding provokes major conformational changes in the
perGK enzyme, whereas ADP binding does not cause further changes in the conformation
of the protein. The results show that although the permutation has no effect on the
nucleotide preference it provokes a change in the substrate binding order that correlates
well with that those observed in the crystal structures. Also, they demonstrate that
during the evolutionary history of the ribokinase superfamily folding topology dictates
the substrate binding order (Fondecyt 1150460).
PC-024
Thrombin Proteolytically Hinders the Antioxidant Activity of Human Ceruloplasmin:
Implications in the Pathogenesis of Rheumatoid Arthritis
Laura Acquasaliente1, Giulia Pontarollo1, Alexiej V. Sokolov2, Simone Tescari1, Vadim
B. Vasilyev2, Vincenzo De Filippis1
1Department of Pharmaceutical and Pharmacological Sciences, University of Padua, 2State
University of Saint-Petersburg
Background: Human ceruloplasmin (CP) is a circulating copper-containing glycoprotein
produced in the liver and first described as a component of alpha2-globulin fraction
of human plasma. CP belongs to the multicopper oxidase family and it is nowadays regarded
as a “moonlighting” protein, because it changes its function according to substrate,
localization and expression. CP plays a key role in copper transport and iron metabolism
and it is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (Kd=130nM), a
major source of oxidants in vivo. The protein is extremely susceptible to proteolysis.
In fact, CP is a structural homolog of coagulation factors V and VIII, that are physiological
substrates of thrombin (FIIa). Interestingly, thrombin participates in both haemostatic
and inflammatory responses: in some focus of inflammation, such as rheumatoid arthritis
(RA), the high activity of FIIa has been documented. It was demonstrated that FIIa
can promote the chemotaxis of neutrophils and monocytes and their adhesion to endothelial
cells, to increase vascular permeability. All these effect are mediated by PAR-1 interaction,
that are abundantly expressed in inflamed rheumatoid synovial tissues. Aims: In this
study the interaction of CP with thrombin was investigated to confirm the participation
of FIIa in “spontaneous” proteolytic degradation of CP. In fact, in vivo the integrity
of CP is essential for its role in the transport or metabolism of copper. Results:
Our results indicated that thrombin cleaves CP in vitro at 481Arg-Ser482 and 887Lys-Val888
bonds, generating a nicked species that retains the native-like fold and the ferroxidase
activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated.
Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically
degraded to a variable extent, with a fragmentation pattern similar to that observed
with FIIa in vitro, and that proteolysis is blocked by hirudin, a highly potent and
specific thrombin inhibitor. We demonstrate that FIIa has intrinsic affinity for CP
(Kd = 60-270 nM), independently of proteolysis, and inhibits CP ferroxidase activity
(KI = 220 ± 20 nM). Mapping of thrombin binding sites with specific exosite-directed
ligands (i.e. hirugen, fibrinogen gamma-peptide) and thrombin analogues having the
exosites variably compromised (i.e. prothrombin, prethrombin-2, alpha-thrombin), reveals
that the positively charged exosite-II of thrombin binds to the negative upper region
of CP, while the protease active site and exosite-I remain accessible. These results
suggest that thrombin can exacerbate inflammation in RA by impairing via proteolysis
the MPO inhibitory function of CP and by competitively inhibiting CP ferroxidase activity.
PC-025
An artificial pathway for isobutene production by direct fermentation: combining metabolic
engineering and protein engineering
Benoit Villiers1, François Stricher1
1Global Bioenergies
The purpose of Global Bioenergies is to develop innovative metabolic pathways for
the production of light olefins from renewable resources, by direct fermentation.
Light olefins (ethylene, propylene, linear butylene, isobutene and butadiene) are
the core of the petrochemical industry. However, microorganisms do not naturally produce
light olefins and no bioprocess to convert renewable resources to these molecules
has been industrialized so far. Global Bioenergies has developed an artificial metabolic
pathway including all the necessary enzymatic reactions from feedstock to isobutene.
The metabolic route leading to isobutene can be divided in three parts, the first
one being the use of natural reactions occurring in the host microorganism. Second,
heterologous natural reactions were introduced into the same host microorganism. Finally,
in contrast with most former approaches, non-naturally occurring reactions as enzymatic
key steps were used, for example the decarboxylation of hydroxyisovaleric acid into
isobutene. Such non-natural critical steps were made possible by taking advantages
of the natural catalytic and substrate promiscuity of exogenous enzymes. Candidate
enzymes are then evolved using systematic, random and semi-rational approaches in
successive rounds in order to reach the desired catalytic efficiency. Since all these
reactions are enzymatic, isobutene can be obtained by direct fermentation, e.g. a
process wherein all the chemical transformations are carried on by the host microorganism.
The scale-up of this process began in November 2014 in a pilot plant installed in
Pomacle-Bazancourt, France, with an annual capacity of 10 tons of oxidation-grade
isobutene. Importantly, production of a volatile compound such as isobutene (and other
light olefins) by direct fermentation presents two major advantages: first, the product
is spontaneously removed from the culture broth, which alleviates the limitations
linked with titer issues. Second, the purification process is considerably easier
and cheaper since no energy consuming methods such as distillation or phase separation
are necessary to purify the end product. For the first time, batches of industrially
produced isobutene from renewable resources have been obtained in the first half of
2015. This isobutene has been in turn converted into isooctane, an additive currently
used to improve gasoline quality, which could also be used as a standalone fuel. A
demonstration plant is planned in Leuna, Germany, with an annual capacity of 100 tons
of polymer-grade isobutene and IBN-One, a joint venture with Cristal Union (4th European
beet processor), has been formed to build and operate the first plant in France converting
renewable resources into isobutene. Finally, while the isobutene process is progressing
towards industrial scale, Global Bioenergies is also developing new artificial metabolic
pathways enabling direct bio-production of Butadiene and Propylene.
PC-026
The development of a coupled enzyme assay to detect isochorismate pyruvate lyase activity
Linda Jäger1, Christian Jäckel1, Peter Kast1, Donald Hilvert1
1LOC, ETH Zürich
Most bacteria and their pathogens exhibit a sensitive iron balance and can acquire
extracellular iron via the secretion of siderophores. In Escherichia coli, iron starvation
leads to the production of isochorismate from chorismate by isochorismate synthase
such as, for example EntC. However, under normal growth conditions the synthesis of
isochorismate is suppressed. This complicates the in vivo analysis of enzymes downstream
of isochorismate, such as the isochorismate pyruvate lyase from Pseudomonas aeruginosa.
Thus far, only a few isochorismate pyruvate lyases have been biochemically and biophysically
characterized. To further the understanding of this group of enzymes a sensitive enzymatic
activity screening assay was lacking. Here we describe the implementation and optimization
of a high throughput screening assay that measures the siderophore salicylate, a fluorescence
product of the isochorismate pyruvate lyase reaction. The assay employs a sequential
enzyme cascade involving the isochorismate synthase EntC, and PchB variants with varying
isochorismate pyruvate lyase activities. Upon salicylate production, a feedback loop
involving an engineered version of the salicylate mediated transcription activator
NahR further enhances the isochorismate pyruvate lyase production and hence salicylate
production. The assay afforded a 500-fold dynamic range between EcCM, a chorismate
mutase of the same AroQ fold lacking isochorismate activity, and PchB with a kcat/Km
of 1.13 x 106 M-1s-1. The assay will greatly facilitate current efforts to identify
novel enzymes with promiscuity for the isochorismate pyruvate lyase activity.
PD-001
3-D interaction homology. Do hydropathic microenvironments dictate amino acid sidechain
conformations?
Mostafa Ahmed1,2, Martin Safo1,2, J. Neel Scarsdale1,3, Glen Kellogg1,2
1Institute For Structural Biology and Drug Discovery, Virginia Commonwealth University,
2Department of Medicinal Chemistry, Virginia Commonwealth University, 3Center For
The Study of Biological Complexity, Virginia Commonwealth University
Protein folding is typically defined in terms of the spatial arrangement of structural
elements, i.e. helices, sheets and loops. We have, however, been developing an alternative
and complementary paradigm based on conserved hydropathic interaction networks within
proteins. These networks can be viewed as environments comprised of a mixture of polar
and hydrophobic interaction fields, and may be the most important factor driving protein
folding. This concept applies even to the lowest structural level within a protein:
the sidechain conformations (or rotamers). Exhaustive statistical analysis of existing
crystallographic structures of proteins showed rotameric preferences and led to the
creation of rotamer libraries frequently used in multiple aspects of structural biology,
e.g., crystallography of relatively low-resolution structures, homology modeling and
biomolecular NMR. However, little is actually known about the forces and factors driving
the preference or suitability of one rotamer over another. In our study, tyrosine
was analyzed since its sidechain has a comprehensive set of hydropathic properties
that made it ideal as a proof of concept residue. Construction of 3D hydropathic interaction
maps of tyrosine residues in our dataset, reveals the environment around each, in
terms of hydrophobic (π-π stacking, etc.) and polar (hydrogen bonding, etc.) interactions.
After partitioning the tyrosines into backbone-dependent bins, a map similarity metric
based on the correlation coefficient was applied to each map-map pair to build matrices
suitable for clustering. Notably, the first bin representing 631 tyrosines, reduced
to 14 unique hydropathic environments with most diversity arising from favorable hydrophobic
interactions with many different residue partner types. Polar interactions for tyrosine
include ubiquitous hydrogen bonding with the phenolic OH and somewhat surprisingly
a handful of unique environments for the tyrosine backbone. All but one of the 14
environments are dominated by a single rotamer, the exception being an environment
defined by a paucity of interactions with the tyrosine ring and as a consequence its
rotamer is indeterminate. This is consistent with it being composed of mostly surface
residues. Each tyrosine residue attempts to fulfill its hydropathic valences and thus,
structural water molecules are seen in a variety of roles throughout these environments.
Alanine was analyzed using the same protocol as well. Having the smallest sidechain
(and small hydropathic interaction maps), alanine allowed us to investigate a significantly
larger database, permitting us to examine the correlation between hydropathic maps
and various structural features. In conclusion, the analysis of hydropathic environments
strongly suggests that the orientation of a residue in a three-dimensional structure
is a direct consequence of its hydropathic environment, which leads us to propose
a new paradigm, interaction homology, as a key factor in protein structure. It is
not the surrounding residues that direct sidechain conformations, but rather the hydropathic
“field” of the surrounding atoms.
PD-002
Folding studies of independent domains of Lysine, Arginine, Ornithine binding protein
(LAO)
Tania Raquel Berrocal Gama1, Jesús Renan Vergara Gutiérrez1, Andrés Escandón Flores1,
Alejandro Sosa Peinado1
1National Autonomus University of Mexico, Faculty of Medicine
Protein folding problem has been addressed from the past 50 years until nowadays,
however, we still can not explain how proteins acquire their native structure from
their amino acid sequence. Different approaches has been taken in order to study protein
folding, for example, the comparative study of folding mechanism between homologues
proteins with high identity of sequence and structure, and the study of independent
regions within a single protein. Previously in our laboratory, thermodynamic and kinetic
folding properties of lysine, ornithine, arginine binding protein (LAO), a 238 amino
acid periplasmic binding protein (PBP), composed by two Rossmann fold domains (one
continuous and the other discontinuous) attached by a hinge region, has been studied.
Even there is a functional research about binding characteristics of histidine binding
proteińs (His J) domains of when expressed independently (Chu, B. 2013); there are
no folding studies in these conditions for this or another PBP´s. It should be noted
that His J shares 70% of sequence identity and tertiary structure (RMSD≈ 1Å) with
LAO. In order to know the folding effect of encoding different domains in the same
poly peptidic chain, as well as its influence in function, we are studying the thermodynamic
and kinetic characteristics of folding of independently expressed lobes of LAO, and
comparing with those of native protein. By now, we expressed and purified the discontinuous
domain. Circular dichroism (CD) and fluorescence intensity spectra show that this
independent domain has primary and tertiary structure. Thermal denaturation has a
single cooperative transition, which indicates this domain is folded. Thermodynamic
analysis of temperature and urea-induced experiments suggest that LAO’s folding characteristics
are not just the addition of those from independent domains. Furthermore, folding
and refolding kinetics suggest the presence of a burst phase intermediate.
PD-003
A hypothesis to reconcile the physical and chemical unfolding of proteins
Guilherme de Oliveira1, Jerson Silva1
1Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
A comprehensive view of protein folding is crucial for understanding how misfolding
can cause neurodegenerative diseases and cancer. When using physical or chemical perturbations,
NMR spectroscopy is a powerful tool to reveal a shift in the native conformation toward
local intermediates that act as seeds for misfolding. High pressure (HP) or urea is
commonly used to disturb folding species. Pressure favors the reversible unfolding
of proteins by causing changes in the volumetric properties of the protein–solvent
system. However, no mechanistic model has fully elucidated the effects of urea on
structure unfolding, even though protein– urea interactions are considered to be crucial.
Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to
develop the “push-and-pull” hypothesis, which helps to explain the initial mechanism
of chemical unfolding in light of the physical events triggered by HP. In studying
MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR
as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall
structural unfolding that occurs upon reaching a threshold concentration of urea.
At subdenaturing concentrations of urea, we were able to trap a state in which urea
is preferentially bound to the protein (as determined by NMR intensities and chemical
shifts); this state is still folded and not additionally exposed to solvent [fluorescence
and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to
pressure denaturation (lower p1/2 and larger ΔVu); thus, urea and HP share concomitant
effects of urea binding and pulling and water-inducing pushing, respectively. These
observations explain the differences between the molecular mechanisms that control
the physical and chemical unfolding of proteins, thus opening up new possibilities
for the study of protein folding and providing an interpretation of the nature of
cooperativity in the folding and unfolding processes.
PD-004
Zinc: A Promoter or Inhibitor for IAPP aggregation?
Feng Ding1, Praveen Nedumpully-Govindan1
1Clemson Unversity
Zinc ions have been found to play an important and yet complex role in human islet
amyloid polypeptide (hIAPP) aggregation, which is associated with β-cell death in
type-II diabetes (T2D). Both concentration-dependent promotion and inhibition of IAPP
aggregation by zinc ions have been observed in vitro. Similarly, at the population
level, both positive and negative correlations were reported between the activity
of a β-cell specific zinc transporter and T2D risk. Zinc ions are able to bind a single
histidine in hIAPP and coordinate the formation of zinc-bound hIAPP oligomers. We
hypothesize that the relative zinc/hIAPP concentration determines the population of
zinc-bound hIAPP oligomers with different molecular weights. We have applied molecular
dynamics (MD) simulations to systematically study the structure and dynamics of a
range of zinc-coordinated hIAPP oligomers, including monomers, dimers, trimers, tetramers,
and hexamers. Our computational results suggest that different zinc-bound oligomers
have distinct aggregation propensities. High-molecular weight oligomers (≥2 peptides)
have higher aggregation propensity than zinc-free and zinc-bound hIAPP monomers at
∼2 mM concentration in silico. Therefore, our results provide a molecular insight
into the complex role of direct zinc binding on hIAPP aggregation. At low zinc/hIAPP
stoichiometry, zinc binding promotes aggregation. As the stoichiometry increases and
zinc ions bind to single hIAPP peptides, the aggregation of hIAPP is inhibited due
to electrostatic repulsion between the charged zinc ions. Our computational study
sheds light on the complex role of zinc on hIAPP aggregation and T2D development.
PD-005
Macromolecular Crowding: From the test tube to the cell
David Gnutt1, Michael Senske1, Simon Ebbinghaus1
1Department of Physical Chemistry II, Ruhr-University Bochum
Biomolecules function in the densely crowded and highly heterogeneous cell, which
is filled up to a volume of 40% with macromolecules [1]. Often, artificial macromolecular
crowding agents are used to mimic these conditions in vitro and the excluded volume
theory is applied to explain the observed effects [2]. However, recent studies emphasize
the role of further contributions aside from a pure volume effect including enthalpic
and solvent effects [3, 4]. We study cosolute effects at high molecular and macromolecular
concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin
in the presence of different concentrations of cosolutes (glucose, dextran, polyethylene
glycol, potassium chloride) [5]. In contrast to the excluded volume theory, we observed
enthalpic stabilization and entropic destabilization forces for all tested cosolutes.
The enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide
solutions of dextran was thereby similar to the effects observed in monomeric glucose.
Further, it remains unclear how such cosolutes reflect the physicochemical properties
of the complex cell environment as a characterization of the in-cell crowding effect
is lacking. Thus, we developed a FRET-based macromolecular crowding sensor to study
the crowding effect in living cells [6]. The averaged conformation of the sensor is
similar to dilute aqueous buffer and cell lysate. We find that the in-cell crowding
effect is distributed heterogeneously and can change significantly upon osmotic stress.
The presented method allows to systematically study in-cell crowding effects and understand
them as a modulator of biomolecular function.
References:
[1] S. Zimmerman, S. Trach, J. Mol. Biol. 1991, 222, 599-620.
[2] H.-X. Zhou, G. Rivas, A. Minton, Annu. Rev. Biophys. 2008, 37, 375-397.
[3] Y. Wang et al., The Journal of the American Chemical Society, 2012, 134, 16614-16618
[4] R. Gilman-Politi and D. Harries, Journal of Chemical Theory and Computation, 2011,
7, 3816-3828
[5] M. Senske, L. Tork, B. Born, M. Havenith, C. Herrmann, S. Ebbinghaus, J. Am. Chem.
Soc. 2014, 136, 9036-9041.
[6] D. Gnutt, M. Gao, O. Brylski, M. Heyden, S. Ebbinghaus, Angew. Chem Int. Ed. 2014,
DOI: 10.1002/anie.201409847
PD-006
Breaking the deleterious effect of urea-unfolded state: consequences for the reversibility
of intermediate species
Angelo Figueiredo1, Sivanandam Veeramuthu2, Oscar Millet2, Eurico Cabrita1
1Faculdade De Ciências E Tecnologia, Universidade Nova De Lisboa, 2CIC BioGUNE, Structural
Biology Unit and Metabolomics Unit
The stability of biomolecules under co-solvent conditions is dependent on the nature
of the co-solvent [1]. This can alter a protein’s properties and structural features
through biomolecular interactions between its functional groups and the co-solvent
molecules. Ionic liquids (ILs) represent a rather diverse class of co-solvents. The
design flexibility of these molten salts is an attractive feature, allowing the properties
of the IL to be tuned to meet the requirements of different applications [2]. Particularly,
the modulation of reaction pathways between folding states, offering possibilities
to control irreversibility in non-native protein aggregation [2]. This has led us
to investigate the impact of ILs as co-solvents with the well-known protein denaturant
urea. Urea is considered to be a non-ionic chaotrope disturbing considerable the grid
of hydrogen bonds with the protein backbone. Urea interacts preferentially with the
protein surface, mainly apolar residues and that dispersion, rather than electrostatic
interactions, is the main energetic contribution to explain the stabilization of the
unfolded state of the protein and the irreversibility of the unfolding process in
the presence of urea [3]. Remarkably, upon addition of choline chloride (ChlCl), 6
M urea-unfolded Im7 refolds and the 1H-15N HSQC NMR spectrum is very similar to the
folded state in 50 mM phosphate buffer, suggesting that Im7 is in a compact, folded
native state in this IL solution. The IL strongly attenuates the denaturation action
of urea on Im7. To better understand how ILs particularly choline chloride can attenuate
the deleterious action of urea we performed 15N chemical shift perturbation, NMR relaxation
dispersion and ZZ-exchange experiments in order to gain access to the physicochemical
mechanism regarding the thermodynamic stability of the species and kinetic barriers
of the folding reaction, particularly on the intermediate species. Our results show
significant changes on conformational dynamics showing that the native state of Im7
is in equilibrium with an intermediate state that is considerable populated at equilibrium.
Comparison of kinetic and thermodynamic parameters describing the equilibrium intermediate
state (EIS) obtained by NMR with previously reported parameters describing the kinetic
intermediate state (KIS) obtained from stopped-flow fluorescence studies shows that
the KIS and EIS are the same species. Thus the Urea:ChlCl mixture induce significant
accumulation of folded intermediates with a high degree of secondary structure content,
containing three of the four helices (I, II and IV) that are docked around a specific
hydrophobic core, whilst non-native docking of these elements create long-lived intermediate
states. The possibility to use ILs for stabilising intermediates open scenarios for
mechanistic studies of protein unfolding/refolding. It is hoped that the results obtained
from the above study will be useful in recommending tailor-made ILs for various applications
in biological systems.
References:
1. P. H. Yancey, et al. Science, 1982, 217, 1214-22.
2. H Weingärtner, et al. Phys. Chem. Chem. Phys., 2012, 14, 415-26.
3. M Candotti, et al. PNAS, 2013, 110, 15, 5933-38
PD-007
Highly concerted domain folding and subunit association of a multidomain multimeric
L asparaginase from hyperthermophile: A mechanistic underpinning of complex protein
folding in extreme environement
Dushyant K. Garg1, Bishwajit Kundu1
1Indian Institute of Technology Delhi
A large body of multidomain protein folding work has been devoted to study monomeric
proteins. How do multidomain multimeric protein fold, avoiding accumulation of stable
intermediate is yet to be studied in detail. Our present study is focussed on understanding
the folding and assembly of the domains of a homodimeric L-aspraginase from a hyperthermophile
Pyrococcus furiosus (PfA). Each monomer of PfA consists of distinct N-and C-terminal
domains (NPfA and CPfA, respectively), connected by a linker. The folding mechanism
of each domain with respect to full length protein was studied by mutating one out
of two tryptophans, one in each domain. Domains were purified and studied individually
to obtain parallel account of the folding of each domain in isolation. Subunit assembly
was studied by analytical size exclusion chromatography (SEC), Multiangle light scattering
and functional activity. Through far UV CD, intrinsic Trp fluorescence and SEC, we
demonstrated that domain folding and subunit association were intimately linked in
full length PfA. Interestingly, en route to its folding there was complete absence
of hydrophobic intermediates as probed by ANS fluorescence. Folding of NPfA was highly
cooperative and, it provides interacting surfaces for CPfA to fold and also facilitates
subunit assembly. The folding cooperativity of isolated domains was very less compared
to the folding cooperativity of their full length counterparts, as indicated by equilibrium
m values. To our surprise, during pH induced denaturation, at pH 2 and 13, the dimer
dissociates into highly hydrophobic folded monomers which readily underwent amyloidogenesis.
We showed that at such extreme conditions, co-operativity in folding process in multidomain
multimeric protein is not solely governed by the folding of individual domains, rather
by concomitant folding and association of domains directly into a quaternary structure.
In other case, where subunit folding occurred prior to association, protein readily
underwent extensive aggregation.
PD-008
GroEL assisted folding of multiple recombinant proteins simultaneously over-expressed
in E.coli
Megha Goyal1, Tapan Kumar Chaudhuri1
1Indian Institute of Technology Delhi
Aggregation prone recombinant proteins very often form inclusion bodies and also exhibits
poor yield of functional protein during in vitro refolding process from chemically
denatured form. Bacterial chaperonin GroEL provides folding assistance to several
proteins, when over-expressed with one of the recombinant proteins. There are instances
that GroEL in presence of few other co-expressed chaperones like DnaJ, DnaK etc provides
better yield of folded protein during homologous and heterologous expression. Considering
the ongoing events in the cells, it is known that molecular chaperone GroEL assists
in the folding of various proteins in the cytoplasm. Hence attempt to fold multiple
recombinant proteins over-expressing simultaneously with the co-expression of chaperones
can be worth trying. This approach may cut down various complexities in the functional
recombinant protein preparation, including time and effective cost. Keeping this view
in mind, folding of two simultaneously expressed aggregation prone proteins, 69 kDa
E.coli maltodextrin glucosidase (MalZ) and 82 kDa yeast mitochondrial aconitase have
been investigated with the co-expression of GroEL and GroES in E.coli cytosol. It
has been previously reported that both the chosen proteins undergo co-expressed GroEL-GroES
assisted folding in E.coli cytosol, when they over-express alone. In this study we
have optimized the over-expression of MalZ and aconitase simultaneously in E.coli.
Further optimisation was carried out to co-express GroEL along with MalZ and aconitase.
Based on the basic philosophy that soluble protein mainly contains folded fraction,
the event of GroEL/ES assisted folding of simultaneously over-expressed proteins,
MalZ and aconitase was monitored through the attainment of soluble proteins under
various sets of conditions such as temperature. The major outcome of the present study
is that, with the GroEL-GroES assistance, the yield of soluble proteins (MalZ and
aconitase) together constitutes higher percentage of folded protein in contrast to
the percent yield when a single protein was over-expressed. Significance of this type
of study relies on the fact that the cells can over-produce higher amount of recombinant
proteins, when multiple over-expression takes place. Not only pushing up cell’s capability
of over-expression, co-expression of GroEL and GroES efficiently assists in the folding
of multiple proteins simultaneously over-expressed in E.coli.
References:
1. Chaudhuri TK, Farr GW, Fenton WA, Rospert S, and Horwich AL. “GroEL/GroES-mediated
Folding of a Protein Too Large To Be Encapsulated” CELL, vol.107, 235-246, (2001).
2. Subhankar Paul, Chanpreet Singh, Saroj Mishra and Tapan K Chaudhuri. “The 69-kDa
Escherichia coli Maltodextrin Glucosidase does not get encapsulated underneath GroES
and folds through trans mechanism during GroEL/GroES assisted folding”. The FASEB
Journal Vol.21 (11) 2874-2885 (2007).
PD-009
Complexity of the Post-transition State Folding of Rd-apocytochrome b562
Shuanghong Huo1, Mojie Duan1,2, Hanzhong Liu1, Minghai Li1
1Clark University, 2Wuhan Institute of Physics and Mathematics, Chinese Academy of
Sciences
Long time-scale computer simulations powered by Anton, a specialized supercomputer,
and the recent advance in force field development open up a new era in the study of
protein folding, allowing us to investigate the folding of medium-sized proteins with
an explicit solvent model. The folding intermediates that exist after the rate-limiting
step are called hidden intermediates. Structures of the mimics of hidden intermediates
of Rd-apocytochrome b562 are resolved by NMR. Based upon the structural features of
the mimics of hidden intermediates, the folding of Rd-apocytochrome b562 after the
rate-limiting step was proposed to follow a specific pathway. We performed molecular
dynamics simulations starting from a hidden intermediate and the native state of Rd-apocytochrome
b562 in explicit solvent, for a total of 37.18 µs mainly with Anton. Markov state
model was used to analyze the simulation results. Besides the experimentally identified
hidden intermediates, we have found other partially unfolded states and misfolded
states and verified that these states occur after the rate-limiting step. Transition-path
theory was employed to calculate the folding flux. To better describe the complexity
of folding, we propose a folding flux network model to replace the specific pathway
model. The experimentally putative hidden intermediates are the constituent nodes
of the network, but the conformational transitions from them to the final state are
not in a sequential manner. Our folding flux network gives a more detailed and complete
description of the complex folding behavior than the simplistic pathway model. Our
observation of the misfolded states occurring near the native state may have implications
in the study of misfolding diseases.
PD-010
Establishment of thermodynamics of protein aggregation using isothermal titration
calorimetry
Tatsuya Ikenoue1, Lee Young-Ho1, Tetsuhei Uenoyama1, Daniel Otzen2, Yuji Goto1
1Institute for Protein Research, Osaka University, 2Interdisciplinary Nanoscience
Center (iNANO), Aarhus University
Amyloid fibrils associated with serious diseases including Alzheimer’s, Parkinson’s,
and prion diseases promoted the challenge of studying protein misfolding, leading
to the development of amyloid structural biology. Amyloid fibrils form in supersaturated
solutions via a nucleation and growth mechanism. Although the structural features
of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics
of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry,
one of the most powerful approaches used to study proteins. Here, with β2-microglobulin,
a protein responsible for dialysis-related amyloidosis, we show direct heat measurements
of the formation of amyloid fibrils using isothermal titration calorimetry (ITC).
The spontaneous fibrillation after a lag phase was accompanied by exothermic heat.
The thermodynamic parameters of fibrillation obtained under various protein concentrations
and temperatures were consistent with the main-chain dominated structural model of
fibrils, in which overall packing was less than that of the native structures. We
also characterized the thermodynamics of amorphous aggregation, enabling the comparison
of protein folding, amyloid fibrillation, and amorphous aggregation. In order to obtain
general thermodynamic properties of protein aggregations, we further investigated
aggregation of glucagon and insulin, two of the most famous amyloidogenic peptide
hormones, using ITC. We also observed characteristic heat of spontaneous amyloid fibrillation
of both proteins after a lag time. Taken all together, we showed that thermodynamic
studies on amyloid fibrillation and amorphous aggregation were indeed possible by
means of ITC-based qualitative and quantitative calorimetric analyses. ITC will become
a promising approach for clarifying the thermodynamic properties of protein aggregates.
The more case studies are required toward the establishment of thermodynamics of protein
misfolding and aggregation
PD-011
Molecular Mechanisms of Cytoplasmic Protein Quality Control
Rivka Isaacson1, Ewelina Krysztofinska1, Santiago Martínez-Lumbreras1, Arjun Thapaliya1
1Chemistry Department, King’s College London
Molecular Mechanisms of Cytoplasmic Protein Quality Control In the crowded environment
of the cell, quality control mechanisms are vital. Proteins that are obsolete or have
strayed from their operative environments must be recycled or rehoused. When hydrophobic
proteins are, for any reason, exposed to the cytosol they are rapidly captured by
protective complexes which shield them from the aqueous surroundings and decide their
fate (by either targeting them to their correct membrane homes or marking them for
degradation by the ubiquitin/proteasome system). The BAG6 holdase is a heterotrimeric
protein complex, comprising BAG6, UBL4a and TRC35, which works closely with the cochaperone
SGTA to triage hydrophobic proteins and pass them along the appropriate pathway. SGTA
also interacts with viral proteins and hormone receptors and is upregulated in numerous
cancer types. These functions require further investigation to determine the scope
of SGTA as a therapeutic target. Our lab has solved the solution structure of the
N-terminal dimerization domain of SGTA and characterised its interaction with two
different ubiquitin-like (UBL) domains in the BAG6 holdase (one from UBL4A and the
other from BAG6 itself) using NMR chemical shift perturbation data and other biophysical
techniques including isothermal titration calorimetry and microscale thermophoresis.
At this meeting I will report on the progress we have made in structurally characterising
further key players that participate in this quality control, with the aim of clarifying
the intricate network of molecular interactions that governs these processes in health
and disease.
Ensemble, ribbon and electrostatics spacefill views of the SGTA dimerization domain
structure. The final panel shows the structure overlaid with its yeast homologue.
References:
Darby, J.F., Krysztofinska, E.M., Simpson, P.J., Simon, A.C., Leznicki, P., Sriskandarajah,
N., Bishop, D.S., Hale, L.R., Alfano, C., Conte, M.R., Martínez-Lumbreras, S., Thapaliya,
A., High, S. & Isaacson, R.L. (2014) PLoS ONE 9(11):e113281
Leznicki, P., Roebuck, Q., Clancy, A., Krysztofinska, E.M., Isaacson, R.L., Warwicker,
J., Schwappach, B. and High, S. (2013) PLoS ONE. 8(3):e59590
Simon, A.C., Simpson, P.J., Goldstone, R.M., Krysztofinska, E.M., Murray, J.W., High,
S. & Isaacson, R.L. (2013) Proc. Natl. Acad. Sci. U.S.A. 110(4):1327-1332
PD-012
Interaction of curcumin analogs with α-Synuclein: Modulation of Aggregation and Toxicity
Narendra Jha1, A. Anoop1, Narasimham Ayyagari1, Pradeep Singh1, I.N.N. Namboothiri1,
Samir Maji1
1Indian Institute of Technology Bombay
Alpha synuclein is a small protein (∼14 kDa) expressed at high levels in dopaminergic
neurons. Fibrillar aggregates of α-synuclein inside the dopaminergic neuron are the
major components of Lewy bodies and Lewy neuritis inclusion, which are considered
as potential hallmark of Parkinson’s disease (PD). Both in vitro as well as in vivo
studies suggest that the soluble, oligomeric forms of α-Syn are the more potent neurotoxic
species, responsible for neuronal injury and death in PD. Therefore, molecules that
inhibit the toxicity of oligomers either by reducing their formation or by converting
their more toxic oligomeric state to less-toxic fibrillar state would be effective
agents for the drug development against PD. Curcumin is one of the Asian food ingredients
which has shown a potential role as therapeutic agent against many neurological disorders
including PD. However, the instability and low solubility makes it less attractive
for use as potential therapeutic agent. The present work focuses on screening of the
compounds similar to curcumin but having better effects on the morphology and toxicity
of oligomeric and fibrillar assemblies of α-Syn, which could be used as therapeutic
agent preferentially over the naturally occurring curcumin. We synthesized and analyzed
the effects of nine compounds, which are structurally similar to curcumin, on different
stages of α-Syn amyloid aggregation. Here, we showed that curcumin and its analogs
accelerate α-Syn aggregation to produce morphologically different amyloid fibrils
in vitro. However, there is no significant effect of curcumin and its analogs on the
secondary structure of preformed α-Syn fibrils. Furthermore, these curcumin analogs
showed differential binding affinities with the preformed α-Syn aggregates, possibly
due to difference in their chemical structures. The present data suggest the promising
role of curcumin analogs in the treatment of α-synucleinopathy disorders.
PD-013
In vitro folding mechanisms determine the forces applied during co-translational folding
Adrian Nickson1, Jeff Hollins1, Ola Nilsson2, Gunnar von Heijne2, Jane Clarke1
1Department of Chemistry, University of Cambridge, 2Department of Biochemistry and
Biophysics, Stockholm University
There is currently much debate as to whether experiments conducted in vitro describe
the folding of proteins in vivo. In particular, it is often suggested that the co-translational
folding of nascent protein chains is dominated by the presence of the ribosome and
associated chaperones, and that folding mechanisms will be affected by the vectorial
nature of translation. Here we use an arrest peptide assay to investigate the co-translational
folding of a number of all-α spectrin domains that exhibit a range of thermodynamic
stabilities and in vitro folding rates. Our unexpected finding is that that the force
exerted on the ribosome by these domains is not related to either the thermodynamic
stability of the domain, or to the folding (loading) rate, but rather to the in vitro
folding mechanism. We infer that the in vitro folding mechanisms of these domains
are unaffected by the presence of the ribosome – even when part of the nascent chain
is retained within the ribosome exit tunnel. There has been much work to date investigating
the intermediates present in stalled translation complexes – but now, for the first
time, we can begin to directly explore the rate limiting transition state in the co-translational
folding of homologous proteins.
PD-014
Can the structure of a protein (H3.1) depend on the treatment of a solvent medium
(explicit vs effective) in a coarse-grained computer simulation?
Ras Pandey1, Barry Farmer2
1University of Southern Mississippi, 2Air Force Research Laboratory
Solvent medium plays a critical role in orchestrating the structure and dynamics of
a protein. In computer simulation modeling of protein structure in a solvent medium,
explicit, implicit, effective-medium, approaches are often adopted to incorporate
the effects of solvation. Because of the complexity in incorporating all atomic and
molecular details, the multiple components, reaching the large-scale, etc. implicit
solvent or effective medium approach is generally more viable than the explicit solvent
methods. Some of the pertinent characteristics such as excluded volume of the solvent
constituents, its concentration, and the underlying fluctuations which may be important
in probing some issues are generally ignored in effective medium or implicit solvent
approaches. Using a coarse-grained approach, we investigate the structure and dynamics
of a protein (a histone, H3.1) in the presence of both effective as well as explicit
solvent media over a range of temperatures with the Monte Carlo simulations. The protein
is represented by a coarse-grained chain of residues whose interactions are described
by knowledge-based residue-residue and hydropathy-index-based residue-solvent interactions.
In effective medium approach, each empty lattice site around the protein structure
acts as a solvent. Only a fraction of lattice sites are occupied by mobile solvent
constituents along with the protein chain in explicit solvent medium. Large scale
simulations are performed to analyze the structure of the protein for a range of residue-solvent
interactions and temperature in both explicit and effective solvent media. We study
a number of local (e.g. solvation and mobility profiles) and global (radius of gyration
and structure factor) physical quantities as a function of temperature. We find that
the response of the radius of gyration of the protein in explicit solvent is different
from that in effective medium solvent. Thus, the presence of fluctuations in explicit
solvent approach have considerable effects on the structure and dynamics of protein
H3.1. Differences due to type of solvent on the response of some of these quantities
as a function of temperature as well as general similarities will be presented.
PD-015
Single-molecule vectorial folding and unfolding through membrane pores
David Rodriguez-Larrea1, Hagan Bayley2,
1University of the Basque Country, Dept. Biochemistry and Molecular Biology, 2Univerisity
of Oxford, Dept. of Chemistry
Protein folding and unfolding in vivo is frequently vectorial. For example, proteins
are synthesized at the ribosome and emerge N-terminal first. As the polypeptide chain
emerges from a 2 nm wide pore is free to fold, interact with partners or misfold1.
In another example, proteins are unfolded at the proteasome by pulling from either
the N or C terminus against a 1-2 nm wide pore, applying a tension on the residues
surrounding the terminus of the protein2. Under this conditions, proteins may behave
differently than when unfolded/refolded with temperature or urea. This may have important
implications, as protein folding and unfolding in vivo is related to both function
and disease. We noticed that vectorial folding is inherently linked to nanometer size
pores. Making use of nanopore technology we developed a method to monitor protein
unfolding during membrane translocation at the single-molecule level3. Briefly, an
oligonucleotide attached at either end of a protein threads a single protein nanopore
inserted in a lipid membrane. In response to an applied membrane potential, the oligonucleotide
pulls the protein through the pore and as it is forced to translocate it unfolds.
Analysing the ionic current we obtain the unfolding pathway and information on the
polypeptide sequence. This methodology has shown that proteins unfold with different
kinetics when pulled from one terminus or the other4. Remarkably, it is also possible
to say whether the protein has been phosphorylated or not, and where5. We have recently
advanced our model system to study protein folding after translocation at the single-molecule
level6. A single-protein molecule was translocated through a pore and forced to translocate
back at predetermined times. We measured the stability of the refolded state at different
times and we obtained the vectorial folding pathway of the protein. Further, we observed
that the protein was capable of co-translocational folding and that this premature
folding contributed to the complete translocation of the protein. Our results show
that nanopore technology applied to proteins can be used to describe the vectorial
folding and unfolding of proteins, providing insight to how these processes may work
in vivo. Further, single-molecule protein sequencing is a possibility that could revolutionise
our knowledge on biological processes.
1. Fedyukina, D. V. & Cavagnero, S. Protein Folding at the Exit Tunnel. Annu. Rev.
Biophys. 40, 337–359 (2011).
2. Lee, C et al. ATP-Dependent Proteases DegradeTheir Substrates by Processively Unraveling
Them from the Degradation Signal. Mol. Cell 7, 627-37 (2001).
3. Rodriguez larrea, D. & Bayley, H. Multistep protein unfolding during nanopore translocation.
Nat. Nanotechnol. 8, 288-95 (2013).
4. Rodriguez larrea, D. & Bayley, H. Protein co-translocational unfolding depends
on the direction of pulling. Nat. Commun. 5, 1–7 (2014).
5. Rosen, C. B., Rodriguez larrea, D. & Bayley, H. Single-molecule site-specific detection
of protein phosphorylation with a nanopore. Nat. Biotechnol. 32, 179–181 (2014).
6. Rodriguez-Larrea, D. & Bayley, H. Single-molecule observation of protein translocation
reveals co-translocational folding of a single domain protein. Submitted.
PD-016
Reversibility and two state behavior in the thermal unfolding of oligomeric TIM barrel
proteins from three bacterial phyla
Sergio Romero Romero1, Miguel Costas2, Adela Rodríguez-Romero3, D. Alejandro Fernández-Velasco1
1Facultad de Medicina, Universidad Nacional Autónoma de México., 2Facultad de Química,
Universidad Nacional Autónoma de México., 3Instituto de Química, Universidad Nacional
Autónoma de México
Thermodynamics studies of oligomeric proteins, which are the dominant protein natural
form, have been often hampered because irreversible aggregation and/or slow reactions
are common. There is not a single report on the reversible equilibrium thermal unfolding
of proteins composed by (β/α)8 barrel subunits, albeit this “TIM barrel” topology
is one of the most abundant and versatile in nature. The eponymous TIM barrel, Triosephosphate
isomerase (TIM) is a ubiquitous glycolytic enzyme that catalyzes the isomerization
of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The unfolding of several
TIMs, mainly of eukaryotic organisms, has been extensively studied. Regarding thermal
unfolding, eighteen TIMs, mainly from eukaryotes, as diverse as Amoebozoa, Euglenozoa,
Ascomycota and Chordata, have been studied. Even though a full thermodynamic characterization
has been hampered by irreversible aggregation and/or the presence of hysteresis in
all of them, the activation parameters that describe the kinetic control of five eukaryotic
TIMs have been reported. We characterized the structure, catalytic properties, association
state and temperature-induced unfolding of the eponymous TIM barrel, Triosephosphate
Isomerase (TIM), belonging to five species representative of different bacterial taxa:
Deinococcus radiodurans (DrTIM), Nostoc punctiforme (NpTIM), Gemmata obscuriglobus
(GoTIM), Clostridium perfringens (CpTIM) and Streptomyces coelicolor (ScTIM). Irreversibility
and kinetic control were observed in the thermal unfolding of NpTIM and GoTIM, while
for DrTIM, ScTIM and CpTIM, the thermal unfolding was found to follow a two-state
equilibrium reversible process, a behavior not observed previously for others TIMs.
Shifts in the global stability curves of these three proteins are related to organismal
temperature range of optimal growth and modulated by variations in maximum stability
temperature and in the enthalpy change at that temperature. Reversibility appears
to correlate with low isoelectric point, the absence of residual structure in the
unfolded state, small cavity volume in the native state structure, low conformational
stability and a low melting temperature. Furthermore, the strong coupling between
dimer dissociation and monomer unfolding may reduce the possibility of aggregation
and favor reversibility. It appears that there is a delicate balance between several
contributions whose concerted interplay is necessary to achieve thermal reversibility
in oligomeric enzymes. Furthermore, the finding that the three reversible proteins
come from organisms from different phyla suggests that unfolding reversibility may
be more common than what is currently known Supported by CONACyT 99857, PAPIIT IN-219913,
Facultad de Medicina-UNAM, and Posgrado en Ciencias Bioquímicas-UNAM.
PD-017
Structural insights into HIV-1 Gag binding to the plasma membrane during virus assembly
Jamil Saad1, Jiri Vlach1, Ruba Ghanam1
1Department of Microbiology, University of Alabama at Birmingham
A critical step in the late phase of human immunodeficiency virus type 1 (HIV-1) infection
is targeting of the virally encoded Gag proteins to the plasma membrane (PM) for assembly.
Prior to assembly, the HIV-1 Gag polyprotein adopts a compact “folded over” conformation
and exists in the monomeric or low-order oligomeric states. Whereas it is established
that the nucleocapsid domain of Gag specifically recognizes motifs in the viral RNA
genome for packaging, there is compelling evidence that the myristoylated matrix (MA)
domain also binds to cellular RNA to prevent premature Gag targeting to intracellular
membranes. Upon transport of Gag to the PM, the interaction of MA with RNA is exchanged
for an interaction of MA with PM components. This molecular switch induces an extended
conformation of Gag, leading to formation of high-order Gag oligomers on the PM. Because
Gag is anchored and therefore captured by its interaction with the available phospholipids,
the intracellular targeting of Gag is likely to be determined by the relative strength
of its interaction with the dominant lipids composing each membrane subcompartment.
The key to understanding this essential molecular switch is elucidating at the molecular
level the interaction of MA with specific PM components. For over two decades, biochemical,
in vivo, in vitro and genetic studies have focused on factors that modulate binding
of retroviral Gag proteins to membranes but only recently the structural and molecular
determinants of Gag assembly have begun to emerge. In addition to the electrostatic
interactions between a highly conserved basic region of MA and acidic phospholipids,
it is now believed that the hydrophobicity of the membrane interior represented by
the acyl chains and cholesterol also play important roles. We employ NMR methods to
elucidate the molecular determinants of Gag binding to the membrane. Our structural
studies revealed that phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), phosphatidylserine
(PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) all interact directly
with the MA domain of Gag. Striking, our data revealed a novel binding mode by which
the 2’-acyl chains of PI(4,5)P2, PS, PE, and PC lipids are buried in hydrophobic pockets
in MA, while the 1’-acyl chains are exposed. These findings led us to propose a trio
engagement model by which HIV-1 Gag is anchored to the PM via the 1’-acyl chains of
PI(4,5)P2 and PS/PE/PC, and the myr group, which collectively bracket a basic patch
projecting towards the polar leaflet of the membrane. In-depth understanding of the
precise role of lipids in virus assembly, and elucidation of the molecular requirements
of Gag-membrane interaction may aid in the development of new antiviral therapeutic
strategies.
PD-018
The structure and function of the Surrogate Light Chain
Natalia Catalina Sarmiento Alam1, Johannes Buchner1
1Department Chemie, Technishe Universität München
The production of functionally antibodies depends on the transition of immature B
cells to mature plasma cells and is tightly linked to several “quality control” check
points. During B cell development, the pre-B Cell Receptor (pre-BCR) is the first
checkpoint which determines the viability and proliferation of the pre-B cell. The
pre-BCR is composed of an immunoglobulin (Ig) heavy chain molecule associated with
an Ig light chain-like molecule called the Surrogate Light Chain (SLC). The SLC is
composed by two proteins λ5 and VpreB which possess a unique region at the N- or C-terminus,
respectively. VpreB lacks a β-strand which is provided by the λ5 protein allowing
the non-covalent interaction essential for formation of the SLC heterodimer. Our understandings
of the molecular mechanism of SLC function and assembly are still at an early stage.
In particular, we do not know how the SLC associates and forms the pre-BCR for the
selection of all heavy chains (HCs). Our study focuses on dissecting the “Fab fragment”
of the pre-BCR to study the effect of the unexpected structural features of the SLC
to gain insight in HC selection. The analysis of the assembly of the SLC revealed
a significant difference between the single domains and the complexes in terms of
stability and assembly. The folding behavior of the CH1 domain in the presence of
the SLC is key for the first quality control mechanism in the endoplasmic reticulum
(ER) prior to surface expression. Our results show that the SLC interacts with CH1
domain in a similar manner to the CL domain. Thus, the folding of the naturally disordered
CH1 domain upon interaction with the SLC releases the HC retention in the ER by BiP.
Taken together, our study provides new insights into the folding and assembly of the
“Fab fragment” of the pre-BCR and paves the way for a detailed mechanistic understanding
of HCs selection by the unique SLC.
PD-019
2D IR spectroscopy reveals a ß-sheet intermediate that dictates the fiber formation
of hIAPP
Arnaldo Serrano1, Ling-Hsien Tu2, Daniel Raleigh2, Martin Zanni1
1Department of Chemistry, University of Wisconsin-Madison, 2Department of Chemistry,
Stony Brook University
Though the 22-29 (SFGAILSS) region of human Islet Amyloid Polypeptide (hIAPP) has
long been known to be crucial for amyloid fiber formation, lack of β-ordering of this
region in structures of the final fiber as determined by both NMA and X-ray has been
puzzling. New evidence now suggests that the FGAIL region forms ordered β structures
only in early intermediates. We present new 2DIR studies on the FGAIL region of hIAPP,
with uniformly 13C 18O labeled amides, along with spectral and kinetic modelling.
Evolution of the peak frequency and 2D lineshape of the labeled region clearly present
a transition from random coil to a stable β sheet, a conclusion which is substantiated
by simulation of the 2D IR spectra. As determined from kinetic modeling, the FGAIL
β-sheet creates a free energy barrier that is the cause of the lag phase during aggregation.
These findings help to rationalize a broad range of previous fragment and mutation
studies as well as provide a mechanism for fiber formation that has self-consistent
kinetics and structures.
PD-020
The temperature dependence of protein stability in living cells
Austin E. Smith1, Larry Z. Zhou1, Annelise H. Gorensek1, Michael Senske2, Gary J.
Pielak1,3,4
1Department of Chemistry, 2Department of Physical Chemistry II, 3Department of Biochemistry
and Biophysics, 4Lineberger Comprehensive Cancer Center
Theory predicts that the cytoplasm, where the concentration of macromolecules can
exceed 300 g/L, alters protein-folding thermodynamics. Specifically, the intracellular
environment is hypothesized to increase the free energy of protein unfolding (i.e.,
increase stability) by reducing the available space, which is a purely entropic effect.
However, in vitro studies under crowded conditions show that enthalpy plays an underappreciated
role. Here, we present the first insight into the enthalpic destabilization of globular
proteins in living cells. Using 19F NMR and the 7-kDa N-terminal SH3 domain of the
Drosophila signal transduction protein drk, we show that the intracellular environment
destabilizes the protein via attractive electrostatic interactions between the cytoplasmic
components and protein’s unfolded ensemble. Our results contradict classic crowding
theory but provide a more complete picture of physiologically relevant protein thermodynamics.
PD-021
Molecular crowding effects on the native and equilibrium intermediate state of a 29
kDa TIM Barrel protein
Ramakrishna Vadrevu1, Jagadeesh Gullipalli1, Rajashekar Kadumuri1, Srividya Subramanian1,
Koushik Chandra2, Hanudutta Atreya3
1Dept. of Biological Sciences, Birla Institute of Technology & Science, 2NMR Research
Centre, Indian Institute of Science, 3Solid State and Structural Chemistry Unit, Indian
Institute of Science
Studies addressing the consequence of crowding that exist in the interior of cells
have reached an interesting stage. Experimental data so far, predominantly from, small
to medium sized proteins are indicating that, in general, natively folded proteins
including, intrinsically disordered, gain structure and stability under conditions
mimicking cell interior. However, on the other hand, a few studies on small proteins
indicate destabilization of the native state. In very few instances, crowding resulted
in compaction and aggregation of the unfolded and partially folded states. Experimental
data on the consequences of cell-like crowding situation on relatively large proteins
with complex folding free energy landscape are absent. alpha subunit of tryptophan
synthase, a 29 kDa TIM barrel protein, provides a unique opportunity to address the
consequence of crowding on the structure and stability of the native state and also
on a partially folded state stable equilibrium intermediate populated in its (un)folding
reactions. In the presence of increasing amounts the most commonly used crowding agent,
Ficoll-70, a non-monotonous increase in the far UV-CD is observed for the native state.
A steady increase up to 250mg/ml Ficoll followed by a decrease in far-UV CD region
is observed, indicating loss of structure at increased concentrations of the crowding
agent. 1H-15N HSQC NMR and fluorescence (FL) spectra confirm the of loss of structure
at higher concentrations of Ficoll-70. Loss of native base line in the urea induced
unfolding reaction monitored by CD and FL clearly confirms the destabilization of
the native state. Similar to the structural changes observed for the native state,
for the equilibrium intermediate state maximally populated at 3 M urea also, non-monotonous
changes in the far UV CD and fluorescence spectra are observed. The highly populated
equilibrium intermediate shows an initial steady increase in the far UV CD signal
followed by a sudden decrease. Our results suggest that the structure of both native
and partially folded states may be affected under crowding conditions.
PD-022
Co-translational protein folding studies of alpha-1 antitrypsin
Conny Wing-Heng Yu1,2, Lien Chu1,2, Xiaolin Wang1,2, Christopher A. Waudby1,2, John
Christodoulou1,2, Lisa D. Cabrita1,2
1Institute of Structural and Molecular Biology, University College London, 2Institute
of Structural and Molecular Biology, Birkbeck College London
Alpha-1 antitrypsin (AAT) is a 44-kDa serine protein inhibitor (serpin), which acts
as an inhibitor of neutrophil elastase within the lungs. During inhibition, the protein
undergoes a dramatic conformational change in which its exposed reactive centre loop
(RCL) is cleaved and inserts into the central A-sheet as an extra beta-strand. This
highly dynamic protein is also susceptible to mutations, resulting in misfolding and
the accumulation of ordered polymers as intracellular inclusions within the endoplasmic
reticulum of hepatocytes, where AAT is synthesized. Despite much knowledge of the
folding and misfolding properties of AAT as an isolated protein, very little is understood
of how AAT acquires its structure during biosynthesis. Like all proteins, the biosynthesis
of AAT takes place on the ribosome, and protein folding occurs in a co-translational
manner as the nascent polypeptide chain emerges from the ribosome’s exit tunnel. This
study aims to develop the biochemical and NMR structural strategies to characterize
the co-translational folding characteristics of AAT as it is being synthesized on
the ribosome. For these studies, we have designed a series of SecM-stalled ribosome
nascent chain complexes (RNC) of AAT of different lengths, which mimics the “snapshots”
of the protein synthesis, capturing the folding process of the nascent chain during
its emergence from the ribosome. Using this library, we have recently developed a
strategy to produce large quantities of the RNCs both in vitro and in vivo within
E. coli, a prerequisite for detailed biochemical and structural studies. Using the
AAT-RNCs, we are developing a suite of biochemical strategies to probe the capacity
for AAT nascent chains to adopt native structure on the ribosome. We have combined
protease inhibition assays, Western blot and native-PAGE analysis to demonstrate that
AAT can fold while bound to the ribosome. In addition, we have employed a cysteine-based
modification “PEGylation” assay to probe low-resolution structural information of
AAT-RNC and this will guide our structural studies by NMR spectroscopy to provide
a detailed understanding of AAT folding on the ribosome at high resolution.
PD-023
PH and Temperature dependent Folding-Unfolding transition of BBL protein under various
Urea concentration
Sangyeol Kim1,2,4, Wookyung Yu3, Bora Kwon3, Seongjun Park2, Iksoo Chang1,2,3
1Research Institute, Daegu Gyeongbuk Institute of Science and Technology, 2Center
for Proteome Biophysics,Daegu Gyeongbuk Institute of Science and Technolo, 3Department
of Brain Science, Daegu Gyeongbuk Institute of Science and Technology, 4Department
of Physics, Pusan National University
Thermodynamic properties of proteins vary with the environmental solvent condition
(temperature, ions, pH, denaturants, etc.). Although the effect of each environmental
factor on proteins has been well studied, the complex effect of more than two environmental
factors was not studied thoroughly. In this study, we investigate the simultaneous
effect of urea denaturation (disruption of non-covalent bonds in proteins) and acid
denaturation (titration of protein residues) on the nature of the folding transition
for 2CYU protein. We performed the molecular dynamics simulations of BBL (PDB code:
2CYU) protein in various urea concentration at 300K. We calculated pH-dependent free
energy landscape using the extended Munoz-Eaton model and described the phase diagram
for the folding transition of BBL at various pH value and urea concentration. We mapped
out the phase diagram of the folding transition of 2CYU, which clarifies the condition
with which it undergoes the cooperative folding transition or the barrierless folding
transition.
PD-024
Biophysical analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol
Paurnima Talele1, Nand Kishore1
1Indian Institute of Technology Bombay
The protein folding process involves one or more distinct populated intermediates.
One such partially folded structure of particular importance observed during protein
folding pathway is molten globule state. The properties of a molten globule state
are intermediate between those of native and unfolded protein molecules. The importance
of studying equilibrium molten globule is in its greater stability and flexible structure
which has been shown to bind a variety of substrates and play a definite role in certain
human diseases via aggregation, misfolding or some other mechanism. A protein must
assume a stable and precisely ordered conformation to perform its biological function
properly. The stability of a protein under specific conditions depends on its interactions
with the solvent environment. Therefore it is essential to understand protein folding
intermediates, protein solvent interactions and protein stabilization. We have made
attempts to thoroughly investigate the formation of stable molten globule state of
the protein induced by alcohol using combination of calorimetric and spectroscopic
techniques. The presentation will cover the topic on biophysical studies on partially
folded states of myoglobin in presence of 2,2,2-trifluoroethanol. The thermal denaturation
of myoglobin was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various
pH values using differential scanning calorimetry and UV-visible spectroscopy. The
most obvious effect of TFE was lowering of the transition temperature with increasing
concentration of TFE up to 1.5 mol•dm-3, beyond which no thermal transitions were
observed. The conformation of the protein was analyzed by a combination of fluorescence
and circular dichroism measurements. At pH 5.0 and 11.0, partially folded states of
myoglobin were confirmed by CD spectroscopy. Quantitative binding of ANS to the TFE
induced molten globule state of myoglobin was studied by using isothermal titration
calorimetry (ITC). The results enable quantitative estimation of the binding strength
of ANS with the molten globule state of myoglobin along with the enthalpic and entropic
contributions to the binding process. The results also suggest occurrence of common
structural features of the molten globule states of proteins offering two types of
binding sites to ANS molecules which has been widely used as a fluorescence probe
to characterize partially folded states of proteins.
PD-025
Structural duality in peptides derived from choline binding repeats
Hector Zamora-Carreras1, Roberto Silva-Rojas1, Beatriz Maestro2, Erik Strandberg3,
Anne Ulrich3, 4, Jesús M Sanz2, Marta Bruix1, M Angeles Jimenez1
1Instituto de Química Fisica Rocasolano (IQFR-CSIC), 2Instituto de Biología Molecular
y Celular, Universidad Miguel Hernández, 3Institute of Biological Interfaces, Karlsruhe
Institute of Technology (KIT), 4dInstitute of Organic Chemistry, Karlsruhe Institute
of Technology (KIT)
The C-terminal domain of the pneumococcal choline-binding protein LytA (CLytA) consists
of six choline binding repeats (CBRs) organized as a ββ-solenoid structure, which
is characteristic of choline binding modules. Each CBR comprises a β-hairpin core
followed by a short linker sequence. Choline molecules are bound between two consecutive
repeats through hydrophobic and cation-π interactions with aromatic side chains. Apart
from its biotechnological applications as an affinity tag for protein immobilization
and purification, CLytA is useful as a model for understanding the folding and stability
of repeat proteins. In this sense, we proposed to get minimal peptides encompassing
the sequence of a single CBR or even only its β-hairpin core able to maintain the
native fold and the ability to bind choline. To that end, we first proceeded to analyze
the peptide comprising the third β-hairpin core, denoted as CLyt3. Based on CD and
NMR data we demonstrate that the peptide CLyt3 conserves its native β-hairpin structure
in aqueous solution, but forms a stable, amphipathic α-helix in detergent micelles
and as well as in small lipid vesicles [1]. Considering the great differences in the
distribution of hydrophobic and polar side chains shown by CLyt3 β-hairpin and α-helix,
we propose that amphipathic structures are stabilized in micelles or lipid vesicles.
This “dual” behavior is the only up-to-now reported case of a micelle-induced conformational
transition between two ordered peptide structures. To check whether other CBR repeats
also undertake β-hairpin to α-helix transition in the presence of micelles, so that
it represents a general tendency ascribed to all pneumococcal choline-binding modules,
we will show new experimental evidences based on CD and NMR structural studies on
peptides derived from the β-hairpin cores of other CLytA repeats, as well as in modified
CLyt3 peptides.
1. Zamora-Carreras et al. (2015). Chemistry-Eur. J. doi:10.1002/chem.201500447 Financial
support from the Spanish MINECO projects no CTQ2011-22514 and BIO2013-47684-R is acknowledged
PD-026
Conformational analysis of fragments of the human Pin1 protein: the influence of charged
amino-acid residues on the ß-hairpin structure
Joanna Makowska1, Dorota Uber1, Wioletta Żmudzińska2, Caterina Tiberi3, Lech Chmurzyński1,
Anna Maria Papini3
1Faculty of Chemistry, University of Gdansk, 2Laboratory of Biopolymer Structure,
Intercollegiate Faculty of Biotechnology, Medical University of Gdansk, 3Dipartimento
di Chimica Organica ‘Ugo Schiff’, Universit'a di Firenze
Continuing our studies of the effect of like-charged residues on protein-folding mechanisms,
in this work, we investigated, by means of NMR spectroscopy and molecular-dynamics
simulations, two short fragments of the human Pin1 WW domain [hPin1(14-24); hPin1(15-23)]
and one single point mutation system derived from hPin1(14-24) in which the original
charged residues were replaced with non-polar alanine residues. Results, for both
original peptide fragments of hPin1 demonstrate the presence of ensembles of structures
with a tendency to form a β-chain reversal.
PD-027
Understanding the biology of Huntington’s disease via the pathogenic huntingtin monomer
Estella Newcombe1, Yasmin Ramdzan1, Ashish Sethi1, Michael Lee2, Dorothy Loo3, Bim
Graham2, James Swarbrick2, Anthony Purcell3, Paul Gooley1, Danny Hatters1
1Department of Biochemistry and Molecular Biology, University of Melbourne, 2Monash
Institute of Pharmaceutical Science, Monash University, 3Department of Biochemistry
and Molecular Biology, Monash University
Huntington’s disease (HD) is caused by an abnormal extension of the polyglutamine
(polyQ) region within exon 1 of the protein huntingtin from typically 25 glutamines
to over 36. Disease onset correlates with the huntingtin misfolding and causing the
formation of aggregates, however recent studies have postulated that pathogenic huntingtin
monomer may form compact structures that are responsible for neuronal toxicity in
HD. We sought to examine the conformation of huntingtin monomers, how polyQ sequence
length affects monomer structure and which protein-binding partners in the cell may
exert a gain-of-toxic mechanism in pathology. Hydrogen-deuterium exchange mass spectrometry
was used to measure the degree of structure in both non-pathogenic (25Q) and pathogenic
(46Q) huntingtin, with results showing that both forms exchanged 79% of potential
NH hydrogen bond donors within 30 seconds (n=3), with little to no further exchange
over the following ten minutes. This result suggested that the pathogenic conformations
are not stabilized by slow exchanging hydrogen bonds. Binding partners to the monomer
were assessed in Neuro2a cell culture by immunoprecipitation and quantitative MS/MS
proteomics approaches after depletion of aggregates by pelleting. Proteins that more
prevalently co-precipitated with pathogenic huntingtin included Fused in sarcoma (Fus),
glycine-tRNA ligase (Gars), peroxiredoxin 6 (Prdx6), phosphatidylethanolamine-binding
protein 1 (Pebp1/Rkip), and histone subunit Hist1H4a, all of which were significantly
enriched by two-fold or greater. RNA-seq analysis indicated that none of these proteins
had altered expression levels, suggesting that the binding interactions are not due
to changes in background abundance. Overall we found that the conformational differences
are subtle, yet are sufficient to generate several specific proteome interactions
that offer clues to a toxic gain-of-function mechanism in pathology. Work is ongoing
to probe the more subtle changes in conformation and the importance of these interactors
to mediating mechanisms of dysfunction.
PD-028
Molecular basis of tyrosinemia and identification of possible pharmacological chaperones
targets
Iratxe Macias1, Arantza Sanz1, Ana Laín1, Oscar Millet1
1CIC bioGUNE
Hereditary tyrosinemia type I is an autosomal recesive disorder caused by deficiency
of fumarylacetoacetate hydrolase (FAH) enzyme. Deficiency of FAH leads to cellular
accumulation of toxic metabolites which include mainly, succinylacetone (SA), maleylacetoacetate
(MAA) and fumarylacetoacetate (FAA) in many body tissues. FAH is mainly expressed
in hepatocytes and renal proximal tubular epithelium. Therefore, liver and kidney
are the two primary organs affected by this disorder, and development of hepatocellular
carcinoma is the major symptom. Missense mutations leads to a loss of enzymatic efficiency
which, in a high number of mutations, correlates with loss of kinetic and thermodynamic
stability of the enzyme. In our ongoing project, we are trying to elucidate the molecular
basis of tyrosinemia by means of biophisical and structural characterization of FAH
wild type along with its mutations. This knowledge should help us design new therapies
based on the identification of pharmacological chaperones that could restore the altered
enzymatic stability of the enzyme. Human FAH wild type and 19 selected mutants were
synthesized and inserted in an expression vector for E. coli. The proteins were purified
in a FPLC and, their thermodynamic and kinetic stability investigated using circular
dichroism. Our preliminary results confirm the loss of termodinamic stability of different
mutants and its variability compared to wild type protein.
PD-029
Repulsion between net charges of subunits during ferritin assembly
Daisuke Sato1, Hideaki Ohtomo1, Atsushi Kurobe1, Satsuki Takebe1, Yoshiteru Yamada2,
Kazuo Fujiwara1, Masamichi Ikeguchi1
1Department of Bioinformatics, Graduate School of Engineering, Soka University, 2JASRI/SPring-8
The organisms have a lot of spherical shell-shaped supermolecules consisting of identical
or distinct subunits (e.g., ferritin, virus capsid, lumazine synthase and encapsulin).
Such multimeric proteins spontaneously assemble into their native structures from
the subunits to acquire the specific functions. However, the assembly mechanism of
such supermolecules has not been understood in detail. Hence, to investigate the assembly
mechanism is biologically important. Escherichia coli non-heme ferritin (Ftn) consists
of 24 identical subunits, which are assembled into a spherical shell-shape with 4/3/2
symmetry. Ftn is able to store iron inside cavity. The subunit includes A-D helices
forming 4-helix bundle, a long BC-loop between B and C-helices and a short E-helix
at the C-terminal. Ftn dissociates into dimers at acidic pH. The dimer was shown to
maintain the native-like secondary and tertiary structures by circular dichroism spectra
and small angle X-ray scattering (SAXS). The acid-dissociated Ftn is able to reassemble
into the native structure when pH increases. To clarify Ftn assembly mechanism, we
performed the stopped-flow time-resolved SAXS (TR-SAXS) experiments. The SAXS profiles
could be acquired every 15 ms after the initiation of reassembly. The initial velocity
calculated from the forward scattering intensity increment was proportional to the
square of the protein concentration, implying that the reaction is second-order. We
propose the sequential bimolecular reaction, in which two dimers bind to form tetramer,
then another dimer attaches to the tetramer to form a hexamer, and so on. The assembly
rate depended on pH and ion strength, indicating that the electrostatic interaction
plays an important role in the assembly reaction. The assembly rate decreased with
increasing pH in the range from 6.0 to 8.0 and increased with increasing NaCl concentration.
This indicates that there are repulsive electrostatic interactions between assembly
units and that they increases with increasing pH from 6.0 to 8.0. A possible interaction
is the repulsion between net charges of dimers since pI of Ftn is expected to be 4.6.
To test this possibility, we made several mutants with different net charges. As mutational
sites, we selected charged residues that are far from the subunit interface. Selected
sites were Glu5, Glu8, Glu12, Glu85 and Glu89. We constructed the mutants with one,
two, three or four Glu -> Gln substitutions of selected sites. The structures of those
mutants were similar to that of wild-type Ftn. If aforementioned hypothesis is correct,
the assembly rate is expected to increase with increasing the number of substitution.
The result agreed well with this expectation and strongly suggested that the electrostatic
repulsion between dimers is an important factor determining the assembly rate of Ftn.
PD-030
Improved modeling of protein unfolding rates and pathways through solvation and modeling
of beta-barrels
Benjamin Walcott1,2, Luís Garreta3, Christopher Bystroff1,2,4
1Department of Biology, Rensselaer Polytechnic Institute, 2Center for Biotechnology
and Interdisciplinary Studies, 3Department of Computer Science, Universdad del Valle,
4Department of Computer Science, Rensselaer Polytechnic Intitute
An understanding of the folding and unfolding pathways of proteins is integral to
improving our ability to associate the structural impact of point mutations and disease
etiology. Information gained here can also be used for protein structure prediction
and design. To model unfolding pathways in proteins we utilize a computational method
called GeoFold. This approach uses recursive hierarchical partitioning of protein
structure and finite elements simulation. GeoFold considers three types of partitioning
operations: translational motion (break), single point revolute joints (pivot), and
rotation around two points (hinge). From these operations, a directed acyclic graph
(DAG) is constructed where nodes correspond to the substructures created by these
operations and the edges represent the operations. For each operation in the DAG,
its dissociation and reassociation rates are determined as a function of solvent-accessible
surface area, hydrogen bonds, voids, and conformational entropy. Finite element simulations
are carried out to simulate the kinetics of unfolding. This model accurately predicts
changes in unfolding pathways due to disulfides in a four-protein case-study, but
it fails to produce a realistic pathway for β-barrel proteins such as green fluorescent
protein (GFP). To better model these barrel proteins, a new partitioning operation
is introduced involving the breaking of all contacts between an adjacent set of β-strands,
called a seam. In addition, to improve the accuracy of kinetic modeling, several updates
have been made to the energy function, including an improved solvation model and a
contact-order-based estimation of the reassociation rates. The predicted unfolding
rates and pathways using this improved GeoFold are compared with experimentally measured
values in KineticDB for proteins with multi-state unfolding kinetics, point mutations,
circular permutations, and engineered disulfides.
PD-031
In the Multi-domain Protein Adenylate Kinase, Domain Insertion Facilitates Cooperative
Folding while Accommodating Function at Domain Interfaces
V. V. Hemanth Giri Rao1, Shachi Gosavi1
1National Centre for Biological Sciences, Tata Institute of Fundamental Research
The presence of multiple domains in a protein can result in the formation of partially
folded intermediates, leading to increased aggregation propensity. This can be reduced
by cooperative, all-or- nothing folding of the multi-domain protein. In good agreement
with ensemble folding experiments, a coarse-grained structure-based model of E. coli
Adenylate kinase (AKE) folds cooperatively. AKE has three domains, NMP, LID and CORE.
We examine the role of the interfaces between these domains in facilitating folding
cooperativity in AKE. Mutants in which these interfaces are deleted exhibit similar
folding cooperativities as wild-type AKE. On closer inspection, we observe that unlike
a typical multi- domain protein in which one domain is singly-linked to its adjacent
domain, NMP and LID are inserted into CORE, i.e. they are both connected to CORE by
two linkers each. We create circular permutants of AKE in which the inserted domains
are converted to singly-linked domains, and find that they fold less cooperatively
than wild-type AKE. Domain insertion in wild-type AKE facilitates folding cooperativity
even when the inserted domains have lower stabilities. The N- and C- termini of NMP
and LID are constrained upon the folding of CORE and this facilitates their folding.
Thus, NMP and LID which undergo large conformational changes during catalysis can
be smaller with fewer stabilizing interactions. In addition, inter-domain interactions
need not be optimized for folding, and can be tuned for substrate binding, conformational
transition and catalysis. Analysis of protein domains using structural bioinformatics
suggests several examples of multi-domain proteins in which domain insertion is likely
to facilitate folding cooperativity.
PD-032
Tuning cooperativity on the free energy landscape of protein folding
Pooja Malhotra1, Jayant Udgaonkar1
1National Centre for Biological Sciences, Tata Institute of Fundamental Research
The mechanism by which a protein explores the free energy landscape during a folding
or unfolding reaction is poorly understood. Determining whether these reactions are
slowed down by a continuum of small (∼ kBT) free energy barriers or by a few large
(> 3 kBT) free energy barriers is a major challenge. In this study the free energy
landscape accessible to a small protein monellin is characterized under native-like
conditions using hydrogen exchange in conjunction with mass spectrometry. Cooperative
and non-cooperative opening processes could be directly distinguished from the mass
distributions obtained in the EX1 limit. Under native conditions, where the native
state is maximally stable, the unfolded state is transiently sampled in an entirely
non-cooperative and gradual manner. Under conditions which stabilize the unfolded
state or destabilize the native state of the protein, the slowest structure opening
event becomes cooperative. The present study provides an understanding of the relationship
between stability and folding cooperativity. It suggests that the cooperative transitions
observed in unfolding reactions maybe a consequence of the changes in the stabilities
of the unfolded state and the transition state. It also provides rare experimental
evidence for a gradual unfolding transition on a very slow timescale.
PD-033
Role of electrostatic repulsion between unique arginine residues on the assembly of
a trimeric autotransporter translocator domain
Eriko Aoki1, Kazuo Fujiwara1, Masamichi Ikeguchi1
1Department of Bioinformatics, Graduate School of Engineering, Soka University
Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family.
The autotransporter consists of an N–terminal signal peptide, an internal passenger
domain and a C-terminal translocator domain. The signal peptide directs to export
across the inner membrane via the Sec system and is cleaved, the passenger domain
is a virulence factor, and the translocator domain (HiaT) is embedded in the outer
membrane. The crystal structure of Hia translocator domain (HiaT) has shown that HiaT
forms a transmembrane β-barrel of 12 β-strands, four of which are provided from each
subunit. The β-barrel has a pore that is traversed by three α-helices, one of which
is provided from each subunit. The protein has a unique arginine residue at 1077.
Arg1077 side chains from three subunits protrude from the β-strand toward the center
of the barrel and are close to each other. These residues seem to have an unfavorable
electrostatic effect on the assembly and decrease the trimer stability. To investigate
the role of this residue on the trimer assembly and stability of HiaT, we replaced
this arginine with the neutral amino acid, methionine (R1077M) or the positively charged
residue, lysine (R1077K), and properties of these mutants were investigated. HiaT
and two mutants were dissociated by formic-acid treatment, and they were able to reassemble
in the presence of the detergent. To measure the time course of trimer reassembly,
amounts of reassembled trimer and monomer were quantified by SDS-PAGE at different
assembly times. Although the neutralized mutation increased the rate of reassembly,
the final amount of reassembled trimer decreased, especially at higher protein concentration.
These suggest that the neutralized mutation cause the incorrect oligomer formation.
The far-UV CD spectrum of reassembled WT HiaT was nearly identical with that of the
native WT HiaT. However, the spectrum of the reassembled R1077M mutant was more intense
that of the native R1077M mutant, although the proportion of trimer was much lower
than that of the WT HiaT. This suggests that the incorrect oligomer has a secondary
structure different from the WT HiaT. R1077K mutant showed assembly properties similar
to those of the WT HiaT. Therefore, the repulsion between positively charged residues
seems to be important for preventing HiaT from misassembly. Similar proximity of arginine
residues is observed for HIV capsid protein, carboxysome shell protein, lumazine synthase
and so on. The electrostatic repulsion between arginine residues may be a general
mechanism for protein assembly.
PD-034
Kurozu Increases HSPA1A Expression and Ameliorates Cognitive Dysfunction in Aged SAM
P8 Mice
Toshiaki Kakimoto1, Hideya Nakano1, Yuji Nakai2, Kazuhiro Shiozaki3, Kohei Akioka4,
Konosuke Otomaru5, Mitsuharu Matsumoto6, Masanobu Nagano7, Yasushi Sugimoto8, Hiroaki
Kanouchi1
1Department of Veterinary Pathobiology, Kagoshima University, 2Institute for Food
Sciences, Hirosaki University, 3Faculty of Fisheries, Kagoshima University, 4Department
of Veterinary Histopathology, Kagoshima University, 5Veterinary Clinical Training
Center, Kagoshima University, 6Department of Veterinary Anatomy, Kagoshima University,
7Sakamoto Kurozu Inc., 8The United Graduate School of Agricultural Sciences, Kagoshima
University
Kurozu is a traditional Japanese rice vinegar. During fermentation and aging of the
Kurozu liquid in an earthenware jar over 1 year, solid residue called Kurozu Moromi
is produced. In the present study, we evaluated whether concentrated Kurozu or Kurozu
Moromi could ameliorate cognitive dysfunction in the senescence accelerated P8 mouse.
Senescence accelerated P8 mice were fed 0.25% (w/w) concentrated Kurozu or 0.5% (w/w)
Kurozu Moromi for 4 or 25 weeks. Kurozu suppressed cognitive dysfunction and amyloid
accumulation in the brain, while Kurozu Moromi showed a tendency to ameliorate cognitive
dysfunction, but the effect was not significant. We hypothesize the effect is caused
by the antioxidant effect of concentrated Kurozu, however, the level of lipid peroxidation
in the brain did not differ in senescence accelerated P8 mice. DNA microarray analysis
indicated that concentrated Kurozu increased HSPA1A mRNA expression, a protein that
prevents protein misfolding and aggregation. The increase in HSPA1A expression by
Kurozu was confirmed using quantitative real-time PCR and immunoblotting methods.
Therefore, the suppression of amyloid accumulation by concentrated Kurozu may be associated
with HSPA1A induction. However, concentrated Kurozu could not increase HSPA1A expression
in mouse primary neurons, suggesting it may not directly affect neurons.
PD-035
Cold Denaturation of Alpha-Synuclein Amyloid Fibrils
Young-Ho Lee1, Tatsuya Ikenoue1, Yasushi Kawata2, Yuji Goto1
1Laboratory of Protein Folding, Institute for Protein Research, Osaka University,
2Department of Chemistry and Biotechnology, Tottori University
Although amyloid fibrils are associated with a number of pathologies, their conformational
stability remains largely unclear. We herein investigated the thermal stability of
various amyloid fibrils. α-Synuclein fibrils, freshly prepared at 37 ºC at neutral
pH, cold-denatured to monomers at 0-20ºC and heat-denatured at 60-110 ºC. Meanwhile,
the fibrils of β2-microglobulin, Alzheimer’s Aβ1-40/Aβ1-42 peptides, and insulin exhibited
only heat denaturation, although they showed a decrease in conformational stability
at low temperature in the presence of chemical denaturants. A comparison of structural
parameters with positive enthalpy and heat capacity changes which showed opposite
signs to protein folding suggested that the burial of charged residues in the fibril
cores contributed to the cold denaturation of α-synuclein fibrils. Reinforced electrostatic
repulsion at low temperatures may promote cold denaturation, leading to a unique thermodynamic
property of amyloid fibrils. We propose that although cold-denaturation is common
to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid
structures may explain amyloid-specific cold denaturation due to the unfavorable burial
of charged side-chains in fibril cores.
PD-036
Key structural differences between TbTIM and TcTIM revealed by thermal unfolding molecular
dynamics simulations
Ángel Piñeiro1, Miguel Costas2, Andrea Gutiérrez-Quezada2
1Dept of Applied Physics, University of Santiago de Compostela, 2Lab. of Biophys.
Chem., Dept of Physical Chemistry, Fac. of Chemistry, UNAM
The thermal unfolding pattern obtained by differential scanning calorimetry for Trypanosoma
cruzi and Trypanosoma brucei triosephosphate isomerase (TcIM and TcTIM) are significantly
different although the crystal structure of both proteins is almost indistinguishable
and the sequences are highly homogolous. In order to explain these differences at
molecular level a set of molecular dynamics simulations were performed at different
temperatures between 400 and 700 K. The obtained trajectories were analyzed in detail
and the residues that showed to be key in the unfolding pathway of each species were
identified. A set of residues that behave significantly different between both proteins
were selected and proposed for mutations. The general aim is to identify the minimum
amount of residue mutations that allow providing TbTIM with the behaviour of TcTIM
and vice versa. Experimental complementary work is also being performed on the same
protein.
PD-037
Repositioning SOM0226 as a potent inhibitor of transthyretin amyloidogenesis and its
associated cellular toxicity
Salvador Ventura1, Ricardo Sant’Anna1, Maria Rosário Almeida2, Natàlia Reixach3, Raul
Insa4, Adrian Velazquez-Campoy5, David Reverter1, Núria Reig4
1Universitat Autònoma de Barcelona, 2Instituto de Biologia Molecular e Celular, ICBAS,
3The Scripps Research Institute, 4SOM-Biotech, 5Universidad de Zaragoza
Transthyretin (TTR) is a plasma homotetrameric protein implicated in fatal amyloidosis.
TTR tetramer dissociation precedes pathological TTR aggregation. Despite TTR stabilizers
are promising drugs to treat TTR amyloidoses, none of them is approved by the Food
and Drug Administration (FDA). Repositioning existing drugs for new indications is
becoming increasingly important in drug development. Here, we repurposed SOM0226,
an FDA-approved molecule for neurodegenerative diseases, as a very potent TTR aggregation
inhibitor. SOM0226 binds specifically to TTR in human plasma, stabilizes the tetramer
in vivo and inhibits TTR cytotoxicity. In contrast to most TTR stabilizers, it exhibits
high affinity for both TTR thyroxine -binding sites. The crystal structure of SOM0226-bound
TTR explains why this molecule is a better amyloid inhibitor than Tafamidis, so far
the only drug in the market to treat the TTR amyloidoses. Overall, SOM0226, already
in clinical trials, is a strong candidate for therapeutic intervention in these diseases.
PD-038
Neurometals as modulators of protein aggregation in neurodegenerative diseases
Sónia S. Leal1, Joana S. Cristóvão1, Cláudio M. Gomes1
1Faculdade de Ciências Universidade de Lisboa - BioISI, FCUL
Protein misfolding and aggregation is a hallmark across neurodegenerative diseases
such as Alzheimer’s disease and Amyotrophic lateral sclerosis (ALS). Since these diseases
are mostly sporadic, the formation of protein amyloids in the nervous system depends
of chemical and biological triggers within the neuronal environment, such as metal
ions [1]. In this communication I will overview the metallobiology of neuronal calcium,
zinc and copper, which are key players in brain function and have altered homeostasis
in most neurodegenerative conditions. Our recent work will illustrate how this allows
establishing molecular mechanisms in neurodegenerative diseases [2-6]. In the pursuit
of this goal, in the last years we have been investigating superoxide dismutase 1
(SOD1), a Cu/Zn metalloenzyme that aggregates in the fatal neurodegenerative disorder
ALS, as a model. In SOD1-ALS cases, this ubiquitous protein selectively aggregates
in motor neurons, implicating a local biochemical factor in the process: interestingly,
Zn2+ and Ca2+ levels are upregulated in the spinal and brain stem motor neurons of
ALS patients, and increased Ca2+ triggers multiple pathophysiological processes which
include direct effects on the SOD1 aggregation cascade [2,3]. Recently we established
that calcium ions promote SOD1 aggregation into non-fibrillar amyloid, suggesting
a link to toxic effects of calcium overload in ALS [4]. We showed that under physiological
conditions, Ca2+ induces conformational changes on SOD1 that increase SOD1 β-sheet
content and decrease SOD1 critical concentration and nucleation time during aggregation
kinetics. We also observed that calcium diverts SOD1 aggregation from fibrils towards
amorphous aggregates. Interestingly, the same heterogeneity of conformations is found
in ALS-derived protein inclusions. We thus hypothesized that transient variations
and dysregulation of cellular Ca2+ and Zn2+ levels contribute to the formation of
SOD1 aggregates in ALS patients [4,5]. In a follow up study we combined experimental
and computational approaches to show that the most frequent ligands for Ca2+ are negatively-charged
gatekeeper residues located in boundary positions with respect to segments highly
prone to edge-to-edge aggregation. Calcium interactions thus diminish gatekeeping
roles by shielding repulsive interactions via stacking between aggregating β-sheets,
partly blocking fibril formation and promoting amyloidogenic oligomers such as those
found in ALS inclusions. Interestingly, many fALS mutations occur at these positions,
disclosing how Ca2+ interactions recreate effects similar to those of genetic defects,
a finding with relevance to understand sporadic ALS pathomechanisms [6]. FCT/MCTES
is acknowledged for grants EBB-BIO/117793/2010 and PTDC/QUI-BIQ/117789/2010 (to CMG)
and BPD (SFRH/BPD/47477/2008 to SSL.)
1. Leal et al (2012) Coord. Chem. Reviews 256(19-20):2253-227
2. Botelho et al. (2012) J Biol Chem 287(50):42233-42
3. Cristovão, et al (2013) Int. J. Mol. Sci. 14(9):19128-45
4. Leal et al (2013) J. Biol. Chem. 288, 25219-25228
5. Leal et al (2015) Metallomics 7(2):333-46 6.Estacio et al (2015) Biochim Biophys
Acta 1854(2):118-26
PD-039
Single-molecule FRET reveals proline dynamics in transmembrane helices
Gustavo Fuertes1,2,3, Ismael Mingarro3, Edward A. Lemke1
1Structural and Computational Biology Unit, European Molecular Biology Laboratory,
2European Molecular Biology Laboratory, 3Department of Biochemistry and Molecular
Biology, University of Valencia
The amino acid proline is well-known by its disorder promoting and helix breaking
properties. Prolines can be accommodated within transmembrane (TM) alpha-helices and
participate in important biological tasks like signal transduction, ligand binding
and helix-helix packing. X-ray crystallography and NMR indicate that proline residues
in membrane proteins induce distortions of the helix geometry to different extents
ranging from small bends to severe kinks. However, such studies provide essentially
a static snapshot of membrane-embedded helices. Therefore, the link between proline
dynamics and function is not completely understood. In this work we have used single-molecule
Förster resonance energy transfer (smFRET) and fluorescence correlation spectroscopy
(FCS) to probe the structure and dynamics of the TM domain of human glycophorin A
(GpA), a widely used model membrane protein for oligomerization studies. A fluorescent
dye pair has been attached to both ends of the membrane-spanning region of GpA, which
allowed monitoring the average distance and distance fluctuations between the attachment
points. Site-specifically double-labeled GpA has been reconstituted into two membrane-mimetic
systems: SDS micelles and phospholipid bilayers assembled into nanodiscs. Using proline-scanning
mutagenesis we have systematically evaluated the impact of proline residues in different
positions along the membrane normal on transmembrane helix length and lateral packing.
Furthermore, we have investigated the distance distribution in TM helices containing
native prolines, namely the insulin receptor and the nesprin protein. Our results
shed light into the relation between proline dynamics and the folding and function
of TM helices.
PD-040
Thermodynamic contributions of specific mutations of L30e protein in the RNA: protein
interface region measured by analytical ultracentrifugation and gel shift assay
Bashkim Kokona1,2, Sara Kim1, Margaret Patchin1, Britt Benner1, Susan White1
1Department of Chemistry, Bryn Mawr College, 2Department of Biology, Haverford College
In Saccharomyces cerevisiae, ribosomal protein L30e acts as an autoregulator by inhibiting
the splicing of its pre-mRNA and translation of its mRNA. The L30e protein-RNA binding
site has been previously studied, revealing a RNA kink-turn motif, which is characterized
by a sharp bend in the phosphodiester backbone due to unpaired nucleotides and internal
tertiary interactions. L30e structural flexibility at the RNA-binding interface makes
such interaction an excellent model to explore the energetics of RNA protein binding.
We made L30e K28A, F85A, and F85W mutants to quantify the thermodynamic contributions
of such interactions to the protein-RNA complex. We used analytical ultracentrifugation
sedimentation equilibrium (SE) and sedimentation velocity (SV) to investigate conformational
changes and protein-RNA binding free energy changes due to mutations. Our computed
changes of binding free energy based on the sedimentation equilibrium experiments
were consistent with the gel shift assay results. In addition, sedimentation velocity
experiments on the L30e wild type indicate that protein-RNA interaction is highly
dynamic and involves conformational changes of the kink-turn RNA induced by L30e protein.
Our results provide new insights on understanding the binding between ribosomal proteins
and their RNA molecules counterpart, which can be used to complement the x-ray structure.
PD-041
Role of a non-native α-helix in the folding of equine β-lactoglobulin
Takahiro Okabe1, Toshiaki Miyajima1, Kanako Nakagawa1, Seiichi Tsukamoto1, Kazuo Fujiwara1,
Masamichi Ikeguchi1
1Department of Bioinformatics, Soka University
Equine β-lactoglobulin is a small globular protein (162 residues). Although ELG adopts
a predominantly β-sheet structure consisting of nine anti-parallel β-strands (A-I)
and one major α-helix in the native state, it has been shown that a non-native α-helical
intermediate accumulates during the burst-phase of folding reaction from the unfolded
state in the concentrated denaturant. To ask whether the non-native helix formation
is important for acquiring the native β-sheet structure, we determined first where
the non-native α-helix is formed. A stable analogue of the burst-phase folding intermediate
was observed at acid pH (A state). The amide hydrogen exchange experiment and proline-scanning
mutagenesis experiment have shown that the non-native α-helix is formed at the region
corresponding to the H strand in the A state. To investigate the role of this non-native
α-helix on refolding reaction of ELG, we constructed several mutant proteins, which
were designed to destabilize the non-native α-helix in the folding intermediate without
perturbation on the native structure. A mutant, A123T, fulfilled this requirement,
that is, A123T showed a native structure similar to that of the wild-type protein,
and largely reduced CD intensity in the A state. Then, the refolding kinetics were
investigated by the CD and fluorescence stopped-flow method. A123T mutation resulted
in reduction of the burst-phase CD intensity, which confirmed that the non-native
α-helix is formed around the H strand region. Subsequent to the burst-phase, four
kinetic phases were observed for A123T and the wild-type protein. Importantly, the
folding rate constants of the four kinetic phases were similar between both proteins.
Furthermore, interrupted refolding experiments demonstrated that the native state
was formed in the two parallel pathways in the two slower phases of the four kinetic
phases. The relative amplitudes of the two pathways were similar between A123T and
the wild-type protein. These results clearly showed that the formation of the non-native
helix has little effect on the folding rates and pathways, and suggested that the
non-native helix formation may not be a severe kinetic trap for protein folding reaction.
PD-042
Impact of the chaperonin CCT in α-Synuclein(A53T) amyloid fibrils assembly
Ahudrey Leal_Quintero1, Javier Martinez-Sabando1, Jose María Valpuesta1, Begoña Sot1
1Centro Nacional de Biotecnología (CNB/CSIC)., 2Centro Nacional de Biotecnología (CNB/CSIC).,
3Centro Nacional de Biotecnología (CNB/CSIC)., 4Centro Nacional de Biotecnología (CNB/CSIC)
and Fundación IMDEA-Nanociencia
CCT is a eukaryotic chaperonin that uses ATP hydrolysis to encapsulate and fold nascent
protein chains. Moreover, it has recently been shown that CCT is able to inhibit amyloid
fibers assembly and toxicity of the polyQ extended mutant of Huntingtin, the protein
responsible of Huntington disease. Although this opens the possibility of CCT being
also able to modulate other amyloidopathies, this has not addressed yet. The work
presented here intends to determine the effect of CCT in the amyloid fibers assembly
of α-Synuclein(A53T), one of the mutants responsible of Parkinson disease. It is demonstrated
that CCT is able to inhibit α-Synuclein(A53T) fibrillation in a nucleotide independent
way, suggesting that this effect is based on binding rather than on active folding.
Furthermore, using deletion mutants and assaying the interaction of CCT with monomers,
soluble oligomers and fibres, it has been possible to unravel the mechanism of this
inhibition: CCT interferes with fibers assembly by interacting with α-Synuclein(A53T)
NAC domain once soluble oligomers are formed, thus blocking the reaction before the
fibers start to grow.
PD-043
Amyloid-like aggregation of Nucleophosmin regions associated with acute myeloid leukemia
mutations
Daniela Marasco1, Concetta Di Natale1, Valentina Punzo1, Domenico Riccardi1, Pasqualina
Scognamiglio1, Roberta Cascella2, Cristina Cecchi2, Fabrizio Chiti2, Marilisa Leone3,
Luigi Vitagliano3
1Department of Pharmacy, CIRPEB: Centro Interuniversitario di Ricerca sui Pepti, 2Section
of Biochemistry, Department of Biomedical Experimental and Clinical Scie, 3Institute
of Biostructures and Bioimaging
Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological
processes and implicated in the pathogenesis of several human malignancies. NPM1 has
been identified as the most frequently mutated gene in acute myeloid leukemia (AML)
patients, accounting for approximately 30% of cases (1). The most frequent human NPM1
mutations lead to variants with altered C-terminal sequences of the C-Terminal Domain
(CTD) that, in its wild form, folds as a three helix bundle. AML modifications lead
to (a) an unfolding of the CTD in the mutated protein and (b) its accumulation in
the cytoplasm due to the loss of nuclear localization sequences with mutations of
Trp290 (mut E) and also of Trp288 (mut A) (2). To gain insights into the role of isolated
fragments in NPM1 activities we dissected the CTD in its helical fragments. Here we
describe the unexpected structural behavior of the fragments corresponding to the
helices H2 and H3 in both wild-type and AML-mutated variants. H2 region shows a remarkable
tendency to form amyloid-like assemblies while only the MutA sequence of H3 region
is endowed with and β-sheet structure, under physiological conditions, as shown by
circular dichroism, Thioflavin T and dynamic light scattering. The aggregates of H2,
are also toxic to neuroblastoma cells, as determined by using the MTT reduction and
Ca2+ influx assays (3). Furthermore the effects of the local context on the different
tendencies to aggregate of H2 and H3 were investigated and appeared to influence for
the aggregation propensity of the entire CTD. Since in AML mutants the CTD is not
properly folded, we hypothesize that the aggregation propensity of NPM1 regions may
be implicated in AML etiology. These findings have implications to elucidate the pathogenesis
of AML caused by NPM1 mutants and aggregation phenomena should be seriously considered
in studies aimed at unveiling the molecular mechanisms of this pathology.
1) Falini, B., et al., 2006, Blood 108, 1999-2005
2) Grummitt, C. G., et al, 2008, J Biol Chem 283, 23326-23332
3) Di Natale C, et al., 2015, FASEB J.,pii: fj.14-269522
PD-044
Transition from α-helix to β-sheet structures occurs in myoglobin in deuterium oxide
solution under exposure to microwaves
Emanuele Calabrò1, Salvatore Magazù1
1Department of Physics and Earth Sciences, University of Messina
We report a resume of our study regarding the effects of microwaves in the range 900-1800
MHz on a typical protein, Myglobin. Previous literature have concerned the effects
on living and in vitro organic systems induced by high frequencies electromagnetic
fields. We have focused our attention on a typical protein, Myoglobin, because proteins
are the simplest organic systems that are fundamentals in organic functions of livings.
Myoglobin is a protein found mainly in muscle tissue of vertebrates, consisting of
a single protein chain with 153 amino acids and one heme group that stores oxygen
in the muscle cells. The physiological importance of Myoglobin is mainly related to
its ability to bind molecular oxygen. In particular, we focused our attention on the
secondary structure of this protein in order to highlight whether exposure to microwaves
unfold the protein producing transitions from α-helix component to β-sheet features.
To this aim Fourier Transform Infrared (FTIR) spectroscopy have been used. The importance
of this study is related to previous literature which indicated that transition from
α-helix to β-sheet structure in a protein can be responsible for aggregation mechanisms
that can lead to neurotoxicity and neurodegenerative disorders that can be considered
as the first step to some pathologies [1-3]. The aggregates consist of fibers containing
unfolded proteins with a prevalent β-sheet structure termed amyloid [4]. In our studies
Myoglobin in deuterium oxide (D2O) solution was exposed for 3 h to mobile phone microwaves
at 900 and 1800 MHz at a power density of 1 W/m2. FTIR spectra were recorded by a
spectrometer Vertex 80v from Bruker Optics, following the protocol accurately described
in [5-7]. FTIR spectroscopy analysis evidenced an increase in intensity of β-sheet
structures and a significant shift to lower frequencies of about 2.5 cm-1 of the amide
I vibration after exposure [8,9]. These results led to conclude that mobile phone
microwaves induce proteins unfolding and formation of aggregates [10, 11].
[1] D. Religa and B. Winblad, Acta Neurobiol Exp, vol. 63, pp. 393-396, 2003.
[2] M. Brzyska, A. Bacia, D. Elbaum, Eur. J. Biochem., vol. 268, pp. 3443-3454, 2001.
[3] M.P. Mattson, Ann N Y Acad Sci, vol. 747, pp. 50-76, 1994.
[4] Kelly, J. W. Curr. Opin. Struct. Biol. 1998, 8, 101–106.
[5] S. Magazù, E. Calabrò, S. Campo, S. Interdonato, Journal of Biological Physics,
vol. 38(1), pp. 61-74, 2012.
[6] S. Magazù and E. Calabrò, The Journal of Physical Chemistry B, vol. 115 (21),
pp. 6818–6826, 2011.
[7] E. Calabrò, S. Condello, M. Currò, N. Ferlazzo, D. Caccamo, S. Magazù and R. Ientile,
Bioelectromagnetics, vol. 34, pp. 618-629, 2013.
[8] E. Calabrò, S. Magazù, Journal of Electromagnetic Analysis & Applications 2(11),
607-617, 2010.
[9] E. Calabrò, S. Magazù, Spectroscopy Letters: An International Journal for Rapid
Communication, Vol. 46 (8), pp. 583-589, 2013.
[10] A.A. Ismail, H.H. Mantch, P.T.T. Wong, Biochim. Biophys. Acta, Vol.1121, pp.
183–188, 1992.
[11] R. Bauer, R. Carrotta, C. Rischel, L. Ogendal, Biophys. J., Vol. 79, pp. 1030–1038,
2000.
PD-045
Investigating the insertion and folding of membrane proteins into lipid bilayers using
a cell free expression system
Nicola Harris1, Kalypso Charalambous1, Eamonn Reading1, Paula Booth1
1Kings College London, Department of Chemistry
Membrane proteins play a vital role in many biological processes, and yet remain poorly
understood as they are frequently unstable in vitro. The goal of this project is to
investigate the insertion and folding of membrane proteins into lipid bilayers, using
a cell free expression system. We have used both E.coli-based cell extracts (S30),
and commercial translation systems (PURExpress) in combination with synthetic liposomes
of defined lipid composition. These studies will aid understanding of cooperative
folding, folding intermediates, and the effects of the lipid bilayer on folding and
insertion. Model E.coli proteins have been investigated, as they can offer important
insights into other proteins, and thus facilitate the further study of more biologically
relevant proteins. It has been found that the rhomboid protease GlpG spontaneously
inserts into liposomes without the aid of an insertase such as SecYEG. This spontaneously
inserted GlpG is functional, and is able to cleave BODIPY-labeled casein, yielding
a fluorescent product. The Major Facilitator Superfamily (MFS) transport proteins
LacY, GalP and GlpT have also been found to insert spontaneously into liposomes. It
has been shown that the lipid composition of the liposomes has an effect on the amount
of protein inserted into the bilayer, with all proteins tested to date preferring
liposomes containing at least 50 mol% DOPG. Ongoing and future work will involve the
use of rare codons to alter the rate of translation, to investigate the effect this
has on the final folded structure of the protein. Preliminary work is also currently
being done into whether the two domains of the MFS family transporters fold cooperatively
or independently, thus aiding understanding into the folding and stability of membrane
transport proteins.
PD-046
Study of rabies virus by Differential Scanning Calorimetry: Identification of Proteins
Involved in Thermal Transitions
Frederic Greco1, Audrey Toinon1, Nadege Moreno1, Marie Claire Nicolaï1, Catherine
Manin1, Francoise Guinet-Morlot1, Frederic Ronzon1
1Sanofi Pasteur, Analytical Research and Development, Biophysical and Biochemical
Study of rabies virus by Differential Scanning Calorimetry: Identification of Proteins
Involved in Thermal Transitions A.Toinon, F. Greco, N. Moreno, M.C. Nicolaï, C. Manin,
F.Guinet-Morlot & F. Ronzon Sanofi Pasteur, Analytical Research and Development, Biophysical
and Biochemical Characterization, 1541 Avenue Marcel Merieux, 69280 Marcy l’étoile,
France Abstract Rabies virus (RABV) is the causative agent of rabies. Rabies remains
an important worldwide health problem that causes a fatal encephalomyelitis [1]. Currently,
rabies in humans is under control in Europe and North America following the use of
efficient vaccines for dogs and wild animals. However, it still kills more than 55,000
people every year mainly in Africa and Asia [2]. Human vaccination prevents infection
with very high efficacy. The vaccine contains an inactivated RABV produced on Vero
cells. RABV is an enveloped, negative single stranded RNA virus which encodes five
proteins, namely the nucleoprotein (N), the phosphoprotein (P), the matrix protein
(M), the glycoprotein (G), and the viral RNA polymerase (L) [3]. The viral envelope
is covered by trimer spikes of G-glycoprotein which is the most significant surface
antigen for generating virus-neutralizing antibodies. Here we illustrate the use of
DSC (Differential Scanning Calorimetry) to identify structural domains or proteins
involved in thermal transitions. The DSC thermogram for intact Beta-propiolactone
inactivated RABV samples in PBS buffer reveals two major thermal transitions with
a Tm respectively at 61°C and 71°C. We have initially focused our investigations on
one of the major proteins encode in RABV, Glycoprotein G [4]. Glycoprotein G contains
disulfide bridges on the ectodomain [6], is sensitive to Bromelain cleavage [5] and
shows reversible conformation changes at low pH [7]. Considering these characteristics,
our results provide evidence on the identity of one thermal transition observed by
DSC.
Keywords:
rabies virus, Differential Scanning Calorimetry, protein unfolding
[1] Hemachudha, T., Laothamatas, J., Rupprecht, C.E., 2002. Human rabies: a disease
of complex neuropathogenetic mechanisms and diagnostic challenges. Lancet Neurology
1
[2] WHO Fact Sheet n°99, updated July 2013
[3] Finke, S., Conzelmann, K.K., 2005. Replication strategies of rabies virus. Virus
Res. 111, 120–131.
[4] Coll, J.M., 1995. The glycoprotein G of Rhabdoviruses. Archives of Virology. 140:827-851.
[5] Gaudin, Y., Tuffereau, C., Segretain, D., Knossow, M., Flamand, A., 1991. Reversible
Conformational Changes and Fusion Activity of Rabies Virus Glycoprotein. Journal of
Virology. 4853-4859
[6] Gaudin, Y., 1997. Folding of rabies Virus Glycoproteein: Epitope Acquisition and
Interaction with Endoplasmic Reticulum Chaperones. Journal of Virology. 3742-3750.
[7] Gaudin, Y., Ruigrok, R., Knossow, M., Flamand, A., 1993. Low-pH Conformational
Changes of Rabies Virus Glycoprotein and Their Role in Membrane Fusion. Journal of
Virology. 1365-1372.
PD-047
Domain swapping of the DNA-binding domain of human FoxP1 is facilitated by its low
folding stability
Exequiel Medina1, Sandro L. Valenzuela1, Cristóbal Córdova1, César A. Ramírez-Sarmiento1,
Jorge Babul1
1Departamento de Biología, Facultad de Ciencias, Universidad de Chile
Domain swapping of the DNA-binding domain of human FoxP1 is facilitated by its low
folding stability Exequiel Medina, Sandro L. Valenzuela, Cristóbal Córdova, César
A. Ramírez-Sarmiento and Jorge Babul Departamento de Biología, Facultad de Ciencias,
Universidad de Chile, Santiago, Chile Protein folding and dimerization (or oligomerization)
are biologically relevant processes when reaching the quaternary structure is required
for function. Proteins that form dimers by exchanging segments or domains of their
tertiary structure with another subunit, the so-called domain swapping phenomenon,
are examples where folding and dimerization are tightly concerted processes. Previous
studies on domain swapping proteins, such as p13suc1 and diphtheria toxin, have shown
that, in general, a high kinetic barrier separates monomers and domain swapped dimers,
and that this barrier can be lowered by promoting protein unfolding and refolding
at high protein concentrations, thus favoring the swapped oligomer. Recent crystal
structures of the DNA-binding domain of several human forkhead box (Fox) proteins
have shown that the P subfamily of these transcription factors (FoxP) can form swapped
dimers. The human FoxP proteins are interesting models of domain swapping, because
mutations of the DNA-binding domain of these proteins are linked to diverse inherited
disorders in humans, such as IPEX and language deficits, and some of these mutations
are located in the hinge region that connects the exchanged segment with the rest
of the protein. Moreover, FoxP1 and FoxP2 have been described to reach monomer-dimer
equilibrium in solution after hours of incubation, suggesting that a low kinetic barrier
separates both species. Using FoxP1 as a model of domain swapping, we analyzed the
temperature and protein concentration effects on the dimer dissociation, obtaining
the free energy change and enthalpy of the process by van’t Hoff analysis (ΔH° of
23.1 kcal•mol-1, ΔS° of 0.082 kcal•mol-1•K-1 and ΔG° at 25°C of −0.95 kcal•mol-1).
These results indicate that the monomer-monomer association is an example of an enthalpy-driven
process. To understand how FoxP1 domains swap without protein unfolding, we performed
equilibrium unfolding experiments using GndHCl as denaturant, showing that the wild-type
protein has a low stability (ΔGU = 6 kcal•mol-1, Cm =3.5 M at 25°C), in contrast to
other domain swapping proteins with high kinetic barriers. We further explore the
domain swapping mechanism of FoxP1 through biased targeted molecular dynamics simulations,
showing that the exchange process can occur by specific local destabilization and
unfolding of the hinge region and helix H3. To further corroborate that the low stability
of wild-type FoxP1 facilitates its domain swapping, we engineered a monomeric version
of FoxP1 through a single-point mutation in the hinge region, which has been previously
described in the literature, and used this protein to visualize the effect of monomer
stability in the dimer formation. Comparison of the folding stability of the monomeric
mutant A39P and wild-type FoxP1 shows that ΔΔGU (mutant- wild-type) is ∼2.5 kcal/mol,
concluding that the ability of FoxP1 to domain swap rapidly can be explained through
its low monomer stability and local unfolding of the exchange region.
Funding: FONDECYT 1130510 and 11140601.
PD-048
Determining the coupled interactions that stabilize the structural framework of the
ß-propeller fold
Loretta Au1, David Green2,3,4
1Department of Statistics, The University of Chicago, 2Department of Applied Mathematics
and Statistics, Stony Brook University, 3Graduate Program in Biochemistry and Structural
Biology, Stony Brook University, 4Laufer Center of Physical and Quantitative Biology,
Stony Brook University
β-propeller proteins are a highly evolved family of repeat proteins that are involved
in several biological pathways, such as signal transduction, cell-cycle modulation
and transcription regulation, through interactions with diverse binding partners,
despite having a similar fold. As for all repeat protein families, there is a consistent
pattern in secondary structure for each repetitive region, in addition to the entire
family. Typically, four to ten propeller blades (each containing four anti-parallel
β-sheets) are arranged in a toroidal shape, thus providing a large binding surface
for ligands or other proteins. About 1% of known proteins adopt this distinctive fold,
and although the requirements for tertiary structure and protein function are fundamentally
encoded in primary structure, this relationship is not fully understood, and addressing
it could provide insight on why the β-propeller fold is common. Many techniques in
comparative sequence analysis can successfully identify amino-acid conservation between
closely related proteins, but molecular interactions between amino acids are often
neglected, and further experimentation is still needed to determine the reasons underlying
conservation. To explore how primary structure can dictate fold and function, we devised
a computational approach to perform large-scale mutagenesis, by adapting the dead-end
elimination and A* search algorithms (DEE/A*), and also leveraged the structural conservation
of each repeating region to understand how sequence variation influences protein fitness,
defined here as a combination of stabilizing and binding interactions. DEE/A* can
evaluate low-energy protein sequences and their corresponding three-dimensional structures,
and we used the β-subunit of a G-protein heterotrimer (PDB: 1GP2, Giα1β1γ2) as a model
system to demonstrate: (1) how the multiple roles of individual amino acids in protein
fitness can be deconvolved, and (2) how epistatic interactions between them can contribute
to structural stability. In doing so, we were able to identify important patterns
in sequence complementarity between repeating regions that cannot be found using sequence-based
methods alone. These results suggest that computational approaches can be used to
determine important protein interactions, and help elucidate the prevalence of β-propeller
proteins in biology.
PD-049
Temperature induced conformational changes of the villin headpiece miniprotein
Stanislaw Oldziej1, Wioletta Żmudzińska1, Anna Hałabis 1
1Intercollegiate Faculty of Biotechnology, UG and MUG
The C-terminal subdomain of the actin-binding protein villin called HP35 (villin headpiece)
has been used as a model protein in a number of studies of protein folding kinetics
and protein folding mechanism [1,2]. The HP35 is a 35 residue miniprotein with an
alpha-helix bundle three-dimensional fold. The goal of our work was to determine conformational
ensemble of polypeptide chain of the investigated miniprotein at a wide range of temperatures
to get detailed information about how protein structure is influenced by temperature.
2D NMR spectra of the title miniprotein were registered at 278, 293 and 313 K. The
three-dimensional structure of the HP35 based on restraints derived from NMR spectra
registered at 278 K is almost identical with structure deposited in the PDB database
in the record 2F4K [2]. At higher temperatures (293 and 313 K) the general shape of
the protein remains unchanged, with well packed hydrophobic core. However, with temperature
increase alpha-helices start to melt. At 313 K structure of the protein remains compact
and in general shape similar to structure observed at 278K, but none of the alpha-helices
could be observed. Results obtained for HP35 protein are in agreement with previous
observation for the Trp-cage miniprotein [3], that with temperature increase regular
secondary structure elements melt first before the break-up of the hydrophobic core
of the protein.
Acknowledgements:
Conference participation for S.O supported by the FP7 project Mobi4Health (grant agreement
no 316094). Calculations were carried out with the use of the resources of the Informatics
Center of the Metropolitan Academic Network (IC MAN – TASK) in Gdańsk, Poland.
1. Kubelka J, Hofrichter J, Eaton WA (2004) The protein folding speed limit Curr Opin
Struct Biol 14:76–88
2. Kubelka J, Chiu TK, Davies DR, Eaton WA, Hofrichter J. (2006) Sub microsecond protein
folding J.Mol.Biol. 359: 546-553
3. Hałabis A, Żmudzińska W, Liwo A, Ołdziej S. (2012) Conformational dynamics of the
trp-cage miniprotein at its folding temperature. J. Phys. Chem. B 116, 6898-6907
PD-050
Comparative equilibrium folding of a membrane transporter within detergent and lipid
environments
Michael Sanders1, Heather Findlay1, Paula Booth1
1Department of Chemistry, King’s college London
Biological membranes provide a selective and chemically sealed barrier for cells.
Transport of ions and small molecules across the membrane is mediated by transporter
proteins and the breakdown of a cell’s ability to produce functionally folded membrane
transport proteins can lead to dysfunction and has been implicated in many diseases1.
However little is known about the processes that govern the misfolding of α-helical
integral membrane proteins, taking into account that these proteins fold and maintain
functional structures within membranes of various organelles. The neurotransmitter
sodium symporter (NSS) protein family is an example of α-helical transporter proteins.
The NSS family encompasses a wide range of prokaryotic and eukaryotic ion-coupled
transporters that regulate the transport of neurotransmitter molecules whose dysfunction
has been implicated in multiple diseases and disorders2. We have investigated the
folding processes of prokaryotic homologue of the NSS family LeuT responsible for
the transport of neurotransmitters and amino acids to the sodium electrochemical gradient.
Previously folding processes of membrane transporters have mainly been characterised
within detergent micelles. However, detergent micelles are not an accurate depiction
of the environment of the membrane bilayer, with this in mind we have also attempted
to investigate folding processes within a bilayer
PD-051
NMR Investigation of pH-induced unfolding of B domain of an Escherichia Coli mannitol
transporter II Mannitol in the bacterial phosphotransferase system
Kim Gowoon1, Yu Taekyung1, Suh Jeongyong1
1Department of Agricultural Biotechnology, Seoul National University
The bacterial phosphotransferase system (PTS) mediates sugar phosphorylation and translocation
across the cytoplasmic membrane. Cytoplasmic B domain (IIB Mtl) of the mannitol transporter
enzyme II Mannitol, a PTS family protein, delivers a phosphoryl group from A domain
to an incoming mannitol that is translocated across the membrane. IIB Mtl is comprised
of a four-stranded ß-sheet and three helices, representing a characteristic Rossmann
fold. We found that the IIB Mtl of Escherichia coli unfolded at a mildly acidic condition.
We made IIB Mtl mutants to investigate the mechanism of the pH-induced unfolding using
NMR spectroscopy. We monitored backbone amide groups and side chain imidazole groups
of histidine residues using 2D HSQC NMR, and pointed out a potential histidine residue
that might be responsible for the unfolding. Histidine residues may be generally important
to the folding stability in response to environmental pH changes.
PD-052
Can site-directed mutagenesis shed light on the refolding pattern of human glucose
6-phosphate dehydrogenase (G6PD)?
Nurriza Ab Latif1,2, Paul Engel1
1Conway Institute, Univerversity College Dublin, 2Faculty of Biosciences and Medical
Engineering, Universiti Teknologi Malaysia
Human glucose 6-phosphate dehydrogenase (G6PD) is the first enzyme involved in the
pentose phosphate pathway (PPP). This oligomeric enzyme catalyses the reaction of
glucose 6-phosphate to form 6-phosphogluconolactone with concomitant reduction of
NADP+ to NADPH. In erythrocytes NADPH is important mainly for protection against oxidative
stress. In connection with its role as the sole source of NADPH, G6PD deficiency commonly
causes haemolytic disease and is known as the most common human enzyme deficiency
globally. Protein folding problems and instability are believed to be the major defects
in the deficient enzymes. In this study, we employed site directed mutagenesis with
hope to give more information on the role of –SH groups in the refolding of human
G6PD. Two mutants were created: 1) one in which all 8 Cys residues were replaced by
Ser and 2) one in which only C13 and C446 were retained. The refolding of recombinant
human G6PD has been studied primarily by measuring the enzyme activity after refolding.
We also used a combination of intrinsic protein fluorescence, ANS (8-anilino-1-naphthalenesulphonic
acid) binding and limited proteolysis to look at the conformational change during
the refolding. The results showed that GdnHCl-denatured recombinant human G6PD wild
type could be refolded and reactivated by rapid dilution technique. Even though, as
recombinants in E. coli, the mutants were well expressed and active, they remained
inactive after attempts were made to refold them in vitro. The methods we applied
may have provided some insights on the refolding pattern of this oligomeric protein,
albeit qualitatively rather than quantitatively.
PD-054
A single aromatic core mutation converts a designed ‘primitive’ protein from halophile
to mesophile folding
Connie Tenorio1, Liam Longo1, Ozan S. Kumru2, C. Russell Middaugh2, Michael Blaber1
1Department of Biomedical Sciences, Florida State University, 2Department of Pharmaceutical
Chemistry, University of Kansas
Experiments in prebiotic protein design suggest that the origin of folded proteins
may have favored halophile conditions. These results are consistent with salt induced
peptide formation which shows that polymerization of amino acids is also promoted
by high salt concentrations. As a result of various origin of life studies, a consensus
on which amino acids likely populated early earth has emerged. These residues were
synthesized by abiotic chemical and physical processes from molecules present in the
surrounding environment. The properties of the consensus set of common prebiotic amino
acids (A,D,E,G,I,L,P,S,T,V) are compatible with known features of halophile proteins,
meaning these proteins are only stable in the presence of high salt concentrations.
The halophile environment, thus, has a number of compelling aspects with regard to
the origin of structured polypeptides. Consequently, a proposed key step in evolution
was, movement out of the halophile regime into a mesophile one commensurate with biosynthesis
of “phase 2” amino acids – including the aromatic and basic amino acids. We tested
the effects of aromatic residue addition to the core of a “primitive” designed protein
enriched for the prebiotic amino acids (A, D, E, G, I, L, P, S, T, V) that required
halophilic conditions for folding. The subsequent results show that the inclusion
of just a single aromatic residue was sufficient for movement to a mesophile folding
environment. Thus, the inclusion of aromatic residues into the codon table could have
conferred key stability to early proteins enabling adaptive radiation outside of a
halophile environment.
PD-055
From Sequence Data to Protein 3D Structure Using Evolutionary Couplings
Robert Fieldhouse1,2, Sikander Hayat1,2, Robert Sheridan1, Debora Marks2, Chris Sander1
1Computational Biology Center, Memorial Sloan Kettering Cancer Center, 2Systems Biology,
Harvard Medical School
Contact prediction methods that rely on sequence information alone, such as EVfold,
can be used for de novo 3D structure prediction and identification of functionally
important residues in proteins. Large multiple sequence alignments of protein families
consisting of evolutionarily related and plausibly isostructural members reveal co-variation
patterns that can be used to identify interactions between pairs of amino acids. We
use a global probability model to disambiguate direct and indirect correlations. Specifically,
we use a maximum entropy approach called pseudo-likelihood maximization (PLM) to distinguish
causation (residue interactions) from correlation (correlated mutations) and compute
evolutionary couplings (ECs). The inferred set of residue interactions can then be
interpreted as physical contacts and used in de novo 3D structure prediction. Furthermore,
the interactions that are inferred can help guide experiments that measure the phenotypic
consequences of protein substitutions, making the method useful for functional studies.
The present work can be divided into three areas: (i) methodological improvements
related to alignment, folding procedure, structure refinement and ranking; (ii) folding
of proteins of known structure for benchmarking and prediction of proteins of unknown
structure; and (iii) focused exploration of specific cases of interest.
PD-056
Developing SHuffle as a platform for expression and engineering of antibodies
Na Ke1, Alana Ali-Reynolds1, Bryce Causey1, Berkmen Berkmen1
1New England Biolabs
SHuffle is a genetically engineered E.coli strain that allows disulfide bond formationin
its cytoplasm with high fidelity. Many proteins containing disulfide bonds have been
successfully expressed in SHuffle. In this study, we expressed, purified and characterized
full-length monoclonal antibody IgG in SHuffle. For the first time, a full-length
IgG can be functionally expressed in the cytoplasm compartment of an E.coli strain.
In order to improve the folding and assembly of IgG, we have investigated the expression
of IgG in various formats and vectors; we have co-expressed chaperones and other helper
proteins with IgG. Several-fold increase in the yield of full-length IgG was observed.
We characterized the SHuffle produced IgG and found it comparable to hybridoma produced
IgG. Optimization of fermentation conditions for a large-scale production is in progress.
We aim to develop SHuffle as an easy, fast, robust platform for antibody engineering,
screening and expression.
PE-001
Experimental and computational studies of the effects of highly concentrated solutes
on proteins: Insights into the causes and consequences of quinary protein structure
and cytoplasmic organization
Luciano Abriata1, Matteo Dal Peraro1
1École Polytechnique Fédérale de Lausanne
Most studies of protein structure and function focus on pure, diluted samples; however,
real-world biochemistry and typical biotechnological applications of proteins take
place in complex media with very high concentrations of solutes (100-400 g/L) of varied
size and chemical nature. On one side, this has recently fostered the study of proteins
in vivo, in cell, or at least in media mimicking the native conditions. On the other
hand, physical chemistry has for a long time studied the general effects of crowded
and viscous conditions on proteins, looking mainly at coarse traits like diffusion
and stability. But the general effects on traits relevant at atomic/residue resolutions
have been less studied, and one fundamental issue remains unsolved: to what extent
are proteins forced into interactions with highly concentrated solutes, and with what
direct consequences? I will present here our ongoing efforts to dissect the fine effects
of high solute concentrations and macromolecular crowding on proteins, based on NMR
experiments and MD simulations, two complementary techniques of high spatial and temporal
resolutions. Our results show that smaller solutes are prone to extensive interactions
with proteins when at high concentrations while large solutes act chiefly through
excluded-volume effects. Overall, we observe location-specific perturbations of a
protein’s surface, its internal dynamics and internal dielectrics, and its hydration,
all very dependently on the solute’s size and chemical nature. Our results support
the growing notion that proteins should be studied in native-like media, adding that
not only macromolecular crowders but also small molecules should be considered in
these studies. Last, the fact that high-concentration conditions affect far more than
a protein’s diffusion rate and stability suggests critical consequences of quinary
protein structure and cytoplasmic organization on the regulation of proteins within
cellular biochemistry.
PE-002
Protein-ligand interactions
Aldona Jelińska1, Anna Lewandrowska1, Robert Hołyst1
1Institute of Physical Chemistry Polish Academy of Sciences
We developed an analytical technique for the study of interactions of ligands (e.g.
cefaclor, etodolac, sulindac) with most abundant blood protein (e.g. bovine serum
albumin) using the Flow Injection Method. The experiments were conducted at high flow
rates (31 cm/s) in a long (>15m), thin (250µm) and coiled capillaries. The compound
of interest (10 µl) was injected into carrier phase, which moved by the Poisseule
laminar flow. At the detection point we measure the concentration distribution of
the analyte. The width of the final profile of the analyte concentration is inversely
proportional to the effective diffusion coefficient of the analyte. From the differences
between the widths of the concentration distribution of free and bound ligand we can
determine value of the association constant.
Acknowledgement:
We thank the National Science Center for funding the project from the funds granted
on the basis of the decision number: UMO-2012/07/B/ST4/01400 (Opus 4).
1. Majcher, A., Lewandrowska, A.; Herold, F.; Stefanowicz, J.; Słowiński, T.; Mazurek,
A.P.; Wieczorek, S.A.; Holyst, R., Anal. Chim. Acta, 855 (2015).
2. Lewandrowska, A.; Majcher, A.; Ochab-Marcinek, A.; Tabaka, M.; Holyst, R., Anal.
Chem. 85 (2013).
3. Bielejewska, A.; Bylina, A.; Duszczyk, K.; Fialkowski, M.; Holyst, R. Anal. Chem.
82 (2010).
PE-003
Carbohydrate Binding Modules. Structural and thermodynamic study
Benjamin Garcia1, Patricia Cano Sánchez1, Siseth Martínez-Caballero1, Romina Rodríguez-Sanoja1,
Adela Rodríguez-Romero1
1Instituto de Química, UNAM, 2Instituto de Química, UNAM, 3Instituto de Química, UNAM,
4Instituto de Investigaciones Biomédicas, UNAM, 5Instituto de Química, UNAM
Carbohydrate binding modules (CBMs), which are defined as contiguous amino acid sequences
within a carbohydrate-active enzyme, have been found in both hydrolytic and non-hydrolytic
proteins and are classified into 71 families, according to their primary structure
similarity. The characterization of CBMs by different methods has shown that these
modules concentrate enzymes on the surface of polysaccharide substrates. It is thought
that maintaining the enzyme in proximity with the substrate leads to more rapid degradation
of the polysaccharide. Therefore, the study of these kinds of modules or domains is
relevant, since they are involved in multiple processes in organisms, like signaling,
defense and metabolism; and some of them are involved in allergenic responses. In
the present work we studied two different models: The first one is a CBM of the family
26 from Lactobacillus amylovorus (LaCBM26) that binds starch These domains are present
in a α-amylase like a repetitive tandem of five modules that are consecutive and do
not present connectors. By means of ITC and using a single recombinant LaCBM26 domain
we determined a Ka = 2.31x104 M-1 for β- cyclodextrin and a Ka = 8.54x104 M-1 for
α-cyclodextrin. When the number of consecutive recombinant modules increased to three
or five tandem modules, the Ka values increased to 106 M-1; however, these constants
did not show an additive or a synergic effect. For these experiments we fitted the
isotherms to different models and used different algorithms. Additionally, we used
circular dichroism in the UV-far region to determine if there existed conformational
changes upon binding of the cyclodextrin molecules to the different tandem modules.
We could only observe slight changes in a positive band centered around 220-240 nm,
which has been explained in terms of π-π; interactions of the aromatic residues at
the binding site. These CBMs have been used as carriers for in vivo vaccine delivery
and affinity tags. The second model is a hevein-like CBM of the family 18 present
in a chitinase-like protein from Hevea brasiliensis (HbCBM18). Hevein is a lectin
from H. brasiliensis that shows a 63% identity with HbCBM18. These CMBs are connected
to the catalytic domain, in proteins such as chitinases, by a linker of approximately
10 residues. In these experiments we used fluorescence techniques to determine the
affinity constants for chitotriose. We previously reported a Ka = 2.08x106 M-1 when
using a HbCBM18 that has a Met residue at the N-terminal region. Besides the aromatic
residues at the binding site, the Met residue also interacts with the ligand, as determined
using crystallographic and docking techniques. The mutant HbCBM18-R5W that does not
have the Met residue showed a Ka of 2.8x104 M-1 with chitotriose, similar to the value
reported for hevein using ITC (Ka 1.42x104 M-1). Interestingly, there exists an isoform
of the HbCBM18 that has a connector between one CBM18 and a half CBM18 (1.5xHbCBM18).
This protein has a Ka of 6.7x105 M-1 with the same ligand.
PE-004
Initiating vesicle formation at the Golgi complex: auto-regulation and protein interactions
govern the Arf-GEFs Gea1 and Gea2
Margaret Gustafson1, J. Chris Fromme1
1Cornell University
Molecular decision-makers play critical roles in the effort to maintain efficient
and accurate cellular functions. In the case of vesicular traffic at the Golgi complex,
the decision to initiate vesicle formation is made by a set of guanine nucleotide
exchange factors (GEFs) that activate the small GTPase Arf1, which is the master controller
for the recruitment of cargos and coat proteins. Saccharomyces cerevisiae possess
three Golgi Arf-GEFs, Gea1, Gea2, and Sec7, which work at distinct sub-compartments
of the Golgi to activate Arf1 only when and where appropriate. In the case of Sec7
at the trans-Golgi network (TGN), this requires a positive feedback loop in which
active Arf1 relieves autoinhibition of Sec7, as well as recruitment to the Golgi membrane
and catalytic stimulation by signaling Rab GTPases. We know far less about the decision-making
process for Gea1 and Gea2, which are responsible for retrograde traffic within the
Golgi and to the endoplasmic reticulum. I have found that both Gea1 and Gea2 can bind
membranes weakly in vitro, an ability which is counteracted by their C-terminal HDS3
domains. In addition, I have discovered membrane recruitment in vitro is aided by
the Rab GTPase Ypt1. However, these interactions cannot fully explain the distinct
localization patterns of Gea1, Gea2, and Sec7, as all three have been shown to be
recruited by Ypt1, which is found throughout the Golgi. My work has revealed that
in addition to the well-established distinct localization from Sec7, Gea1 also occupies
different Golgi compartments from Gea2, so specific signals must exist which help
the GEFs decide where to go. My current efforts focus on understanding the roles of
the other domains of Gea1 and Gea2, identifying the signals which send them to different
parts of the Golgi, and unraveling the different roles they play in vesicle trafficking
pathways.
PE-005
Sequence variation in Archaea through diversity-generating retroelements
Sumit Handa1, Blair G Paul2, Kharissa L Shaw1, David L Valentine2, Partho Ghosh1
1Department of Chemistry and Biochemistry, University of California San Diego, 2Marine
Science Institute, University of California
Protein diversification is an essential tool for the survival and evolution for various
species. Diversity-generating retroelements (DGR) in bacteria is known to generate
massive variation in DNA through an error prone reverse transcriptase and retrohoming,
which leads to variation in protein sequence. Recent discovery of DGRs in intraterrestrial
archaeal systems have opened an opportunity to study this massive sequence variation
in third domain of life (Paul BG, et al. Nat. Comm.) Here, we present the first crystal
structure of variable protein from archaea with ligand-binding pocket is surface exposed.
Also, it has conserved C-type lectin (CLec) fold, as shown by previous work on variable
proteins, major tropism determinant (Mtd) and Treponema variable protein A (TvpA)
which bind ligands through the CLec fold. Despite weak sequence identities (10-15%)
among these variable proteins, CLec fold was found to be conserved. This variable
ligand-binding site for archaea variable proteins can potentially generate 1013 variants.
PE-006
Studies of JMJD4-catalyzed oxidative modifications of eukaryotic release factor 1
Suzana Markolovic1, Ivanhoe K. H. Leung2, Mathew L. Coleman3, Timothy D. W. Claridge1,
Sarah E. Wilkins1, Christopher J. Schofield1
1Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 2School
of Chemical Sciences, University of Auckland, 3School of Cancer Sciences, University
of Birmingham
Protein synthesis is a dynamic process mediated by a variety of proteins and enzymes.
Recent studies have shown that hydroxylation is a key post-translational modification
involved in translation termination. In particular, the Fe(II)- and 2-oxoglutarate-
dependent oxygenase, Jumonji domain-containing 4 (JMJD4), regulates translation termination
via the carbon 4 hydroxylation of an invariant lysine residue, K63, of the eukaryotic
release factor, eRF1. In eukaryotes, translation termination is mediated by a release
factor complex that includes eRF1. eRF1 is comprised of three domains, and it is responsible
for recognizing stop codons in mRNA transcripts before triggering polypeptide release
from the ribosome. The lysine residue hydroxylated by JMJD4 falls within the N-terminal
domain and more specifically within the highly conserved NIKS motif. This motif has
been identified by cross-linking and mutagenesis studies to play an essential role
in stop codon recognition. While hydroxylation of K63 by JMJD4 has been found to increase
translational termination efficiency, the exact molecular mechanism by which hydroxylation
influences termination remains unclear. This work aims to understand how hydroxylation
of eRF1 affects translation termination by exploring the effect of hydroxylation on
the structure, dynamics, stability, and binding of the N-terminal domain of eRF1 (eRF1-N)
using mass spectrometry, protein NMR spectroscopy, circular dichroism and differential
scanning fluorimetry. In our efforts to understand the effect of hydroxylation, an
additional JMJD4-catalyzed modification, characterized by a +30 Da mass shift on K63,
was identified in vitro. The effect of this modification on eRF1 was similarly explored.
Our findings suggest that hydroxylation has no effect on the in-solution NMR structure
of eRF1-N, which experiences chemical shift changes localized to the target lysine
residue. Correspondingly, there are no significant differences in secondary structure
content between wild type and hydroxylated eRF1-N. Hydroxylation was also found to
have no effect on protein stability or dynamics. Interestingly however, the +30 Da
modification appears to cause more significant chemical shift changes dispersed beyond
the NIKS motif. This suggests a more global effect on the in-solution NMR structure
despite the little differences observed in protein dynamics and secondary structure
content. The +30 Da modification was also found to have a destabilizing effect on
eRF1-N. Neither hydroxylated nor +30 Da modified eRF1-N exhibited differences in rRNA
binding. While hydroxylation of eRF1 was found to have little effect on protein structure,
dynamics, stability, or binding, the +30 Da modification has marked effects on protein
structure and stability. Such differences suggest that this modification has the potential
to play an important role in translation.
PE-007
Functional and structural analysis of a GH20 ß-N-acetylglucosaminidase from the marine
bacterium Vibrio harveyi
Piyanat Meekrathok1, Arthur T. Porfetye2, Marco Bürger2, Ingrid R. Vetter2, Wipa Suginta1
1Biochemistry-Electrochemistry Research Unit, Suranaree University of Technology,
2Max Planck Institute of Molecular Physiology
Vibrio harveyi β-N-acetylglucosaminidase (so-called VhGlcNAcase) is a new member of
the GH20 glycoside hydrolase family responsible for the complete degradation of chitin
fragments, with N-acetylglucosamine (GlcNAc) monomers as the final products. However,
the 3D structure of GlcNAcase is still unknown. In this study, crystal structure and
function of GlcNAcase were investigated based on protein crystallography. Size-exclusion
chromatography and the Native-PAGE were employed to verify the protein state of GlcNAcase
in a native form and the acidic active-site residues were mutated using site-directed
mutagenesis method. The effects of mutations on the binding and hydrolytic activities
were studied by enzyme kinetics. To provide a structural basis of GlcNAcase, the wild-type
enzyme was crystalized at 293 K using a solution containing 0.1 M sodium acetate pH
4.6 and 1.3 M sodium malonate and recorded X-ray data. The wild-type enzyme was crystallized
within 3 days in the monoclinic crystal form, belonging to space group P21, with unit-cell
parameters a = 90.2, b = 130.7, c = 98.5 Å. The crystal structures of V. harveyi GlcNAcase
were solved and refined to highest resolution of 2.4 Å. Structural investigation revealed
that GlcNAcase comprises three distinct domains, designated as the N-terminal carbohydrate-binding
domain, the α+β topology domain and the TIM-barrel catalytic domain. The substrate
binding groove of GlcNAcase is a small pocket, which is suitable to accommodate a
short-chain chitooligosaccharide. Kinetic analysis revealed that a group of the adjacent
D303-H373-E438 showed a significantly decreased activity as compared with the wild-type
enzyme, and these residues might be important for enzyme catalysis.
PE-008
Silencing the molecular timekeeper in human cancer
Alicia Michael1, Stacy Harvey1, Patrick Sammons1, Amanda Anderson2, Hema Kopalle1,
Alison Banham2, Carrie Partch1
1University of California - Santa Cruz, 2University of Oxford
The circadian clock coordinates temporal control of physiology by regulating the expression
of at least 40% of the genome on a daily basis.1 Disruption of circadian rhythms through
environmental stimuli (e.g. light at night) or genetic means can lead to the onset
of diseases such as: diabetes, cardiovascular disease, premature aging and cancer.2–5
The circadian clock orchestrates global changes in transcriptional regulation via
the bHLH-PAS transcription factor CLOCK:BMAL1. Pathways driven by other bHLH-PAS transcription
factors have a homologous repressor that modulates activity on a tissue-specific basis,
but none have been identified for CLOCK:BMAL1. We discovered that the cancer/testis
antigen PASD1 fulfills this role to suppress circadian rhythms. PASD1 is evolutionarily
related to CLOCK and interacts with the CLOCK:BMAL1 complex to repress transcriptional
activation. Furthermore, deletion of one region, highly conserved with CLOCK Exon
19, alleviates repression by PASD1 to suggest that it utilizes molecular mimicry to
interfere with CLOCK:BMAL1 function. Structural and biochemical studies of the direct
interaction of PASD1 with the CLOCK:BMAL1 complex using recombinant protein expression
and biophysical techniques are currently underway. As a cancer/testis antigen, expression
of PASD1 is natively restricted to gametogenic tissues but can be upregulated in somatic
tissues as a consequence of oncogenic transformation. Reducing PASD1 in human cancer
cells significantly increases the amplitude of transcriptional oscillations to generate
more robust circadian rhythms. Our work suggests that mechanisms to suppress circadian
cycling can be hard-wired in a tissue-specific manner and our data show that they
can be co-opted in cancer cells to attenuate clock function.
1. Zhang, R., Lahens, N. F., Ballance, H. I., Hughes, M. E. & Hogenesch, J. B. A circadian
gene expression atlas in mammals: Implications for biology and medicine. Proc. Natl.
Acad. Sci. 111, 16219–16224 (2014).
2. Filipski, E. & Lévi, F. Circadian disruption in experimental cancer processes.
Integr. Cancer Ther. 8, 298–302 (2009).
3. Jeyaraj, D. et al. Circadian rhythms govern cardiac repolarization and arrhythmogenesis.
Nature 483, 96–99 (2012).
4. Kondratov, R. V, Kondratova, A. A., Gorbacheva, V. Y., Vykhovanets, O. V & Antoch,
M. P. Early aging and age-related pathologies in mice deficient in BMAL1, the core
componentof the circadian clock. Genes Dev. 20, 1868–1873 (2006).
5. Marcheva, B. et al. Disruption of the clock components CLOCK and BMAL1 leads to
hypoinsulinaemia and diabetes. Nature 466, 627–631 (2010).
PE-009
New insights into the interaction between IQGAP1 and Rho family proteins
Kazem Nouri1, Mohammad Reza Ahmadian1
1Medical faculty of the Heinrich-Heine University
The scaffolding protein IQGAP1 participates in various cellular functions such as
cell-cell adhesion, cell polarization and migration, neuronal motility, and tumor
cell invasion by binding to target proteins, including Rac1 and Cdc42, two members
of the Rho family. To better understand the molecular basis of these interactions,
we utilized in this study a novel time-resolved fluorescence spectroscopy to determine
individual rate constants for IQGAP1 interaction with fourteen different Rho proteins.
The results indicated that IQGAP1 binds among Rho proteins selectively to Rac- and
Cdc42-like proteins only in a GTP-dependent manner. Moreover, the interaction of Rho
proteins with the C-terminal half of IQGAP1 (GRD-C), shorter fragment contains GRD-GBD,
only the GRD and also GRD-GBD with single and double phosphomimetic mutations S1441E
and S1443D was performed. Obtained results showed that, when both GRD and GBD are
existing, fluorescence changes is detected but for GRD alone or in the case of S1443D
or S1441E/S1443D no change was observed, suggesting that GBD and specifically, cysteine
1443 is critical for this interaction. Furthermore, fluorescence polarization results
showed that the GRD-C interact with Cdc42 and Rac1 but not with RhoA, and interestingly
the GRD domain showed similar behavior, but with 10 to 15 folds lower affinity as
compared with the GRD-C. Consistent with this, a GDP-bound form of Cdc42 showed interaction
with both GRD and the GRD-C in quiet comparable affinities. At last, competition experiments
utilizing interacting partners of Rac1, e.g. Tiam1, p50RhoGAP, Plexin-B1, p67phox,
PAK1 and RhoGDIα, along with structural analysis, revealed two negative charged areas
on the surface of Rho- and Rnd-like proteins, which might explain their inaccessible
interaction with IQGAP1. The overlapping binding site of Cdc42 and Rac1 on the surface
of IQGAP1 together with the kinetic details of the selective interaction of IQGAP1
with Rac- and Cdc42-like proteins suggests that these interactions are most likely
mediated via the same mechanism.
PE-010
Structural Characterization of the Tumor Suppressor ING5 as a Bivalent Reader of Histone
H3 Trimethylated at Lysine 4
Georgina Ormaza Hernandez1, Jhon Alexander Rodríguez1, Alain Ibáñez de Opakua1, Nekane
Merino1, Maider Villate1, Tammo Diercks1, Pietro Roversi2, Adriana L. Rojas1, Francisco
J. Blanco1,3
1CIC bioGUNE, Structural Biology Unit, 2Oxford University, Department of Biochemistry,
3IKERBASQUE, Basque Foundation for Science
The INhibitor of Growth (ING) family of tumor suppressors consists of five homologous
proteins that regulate the transcriptional state of chromatin by recruiting histone
acetyl transferase and histone deacetylase complexes to sites with histone H3 trimethylated
at K4 (H3K4me3)1. This modification is recognized by the Plant HomeoDomain (PHD) present
at the C-terminus of the five ING proteins2. ING4 dimerizes through its N-terminal
domain, with a symmetric antiparallel coiled-coil structure3, making it a bivalent
reader of the H3K4me3 mark. ING5 is highly homologous with ING4, but forms part of
a different histone acetyl transferase complex1. Here, we show that ING5 is also a
dimer and thus a bivalent reader of the H3K4me3 mark. However, the crystal structure
of the N-terminal domain of ING5 shows an asymmetric dimer, different from the homologous
ING4 domain. Our NMR data (backbone assignment and paramagnetic relaxation effects)
and SAXS data indicate that the structure of the N-terminal domain of ING5 in solution
is similar to ING4, suggesting that the crystal structure of ING5 is likely a crystallization
artifact. Three point mutations in the N-terminal domain of ING5 have been described
in oral squamous cell carcinoma: Q33R, I68V, and C75R4. We have found that the N-terminal
domains of the three mutants are dimeric coiled-coils but with different stability,
as measured by thermal denaturation. While the Q33R mutant is as stable as the wild
type, the I68V and C75R mutants are strongly destabilized, suggesting a role in cancer
development at least for these two mutants.
References:
[1] Y. Doyon et al. (2006) Mol Cell 21, 51-64.
[2] P.V. Peña et al. (2006) Nature 442, 100-3.
[3] S. Culurgioni et al (2012) J. Biol. Chem. 287(14),10876-84
[4] B. Cengiz eta al (2010) Int J Cancer 127(9), 2088-2094.
PE-011
ABELSON TYROSINE KINASE, A NEW ENZYME TARGET FOR ALZHEIMER’S DISEASE: EXPLORING MULTIPLE
E-PHARMOACOPHORE MODELING, VIRTUAL SCREENING, SELECTIVITY ASSESSMENT FOR POTENTIAL
INHIBITORS
Ravichand Palakurti1, Ramakrishna Vadrevu1
1Department of Biological Sciences, BITS-PILANI HYDERABAD CAMPUS
Efforts so far, to combat Alzheimer’s disease (AD) have focused predominantly on inhibiting
the activity of enzyme(s) that are responsible for the production of the main causative
beta amyloid forming peptide. However, the inherent complexity associated with the
network of pathways leading to the progress of the disease may involve additional
targets for designing effective therapies. Recent experimental findings have identified
Abelson’s Tyrosine Kinase (c-Abl), a non-receptor kinase involved in a variety of
cellular functions as a new target for AD. In the present study we employed energy
optimized multiple pharmacophore modeling strategy from multiple c-Abl structures
bound with ligands in the inactive ATP binding conformation. Virtual screening followed
by docking of molecules from ChemBridge_CNS database, and Maybridge databases resulted
in the identification of 15 best scoring molecules. Based on docking score and selectivity
assessment and druggability parameters, four out of the 15 molecules are predicted
to show increased specificity for c-Abl in comparison to closely related kinases.
Given the implied role of c-Abl not only in AD but in Parkinson’s disease, the identified
compounds may serve as leads to be developed as effective neurotherapeutics.
PE-012
The Role of Syndecans in Melanocortin Signaling and Energy Balance
Rafael Palomino1, Glenn Millhauser2, Pietro Sanna2
1University of California Santa Cruz, 2The Scripps Research Institute
The central melanocortin system is recognized as a key regulator of energy balance
and appetite. The hypothalamic melanocortin receptor, MC4R, is a G-protein coupled
receptor that is antagonized by the peptide ligand, agouti-related peptide (AgRP),
leading to increased feeding and weight gain. While much research has gone into how
this ligand exerts its effects at the receptor, less is known regarding non-melanocortin
components of the pathway. Syndecan-3, a heparan sulfate proteoglycan, has previously
been implicated in potentiating AgRP antagonism, however details of this interaction
are unclear. This work aims to investigate the role of syndecans at both a molecular
level and in vivo. We hypothesize that AgRP binds the glycosaminoglycan (GAG) components
of syndecans, and that this interaction increases the local concentration of the peptide
near MC4R. Furthermore, we have previously shown that designed positive charge mutations
to AgRP lead to increased in vivo efficacy that is independent of MC4R activity, and
we hypothesize that this is due to greater affinity for the negatively charged GAGs.
Using isothermal titration calorimetry we have shown tight binding between AgRP and
heparan sulfate, the major GAG component of syndecan-3, and this affinity is strengthened
by additional peptide positive charge. Through NMR, we see that both positively charged
and polar residues are necessary for binding various heparan sulfate polymers. These
data implicate a specific region of AgRP that is not required for MC4R binding as
being necessary in its role as a heparan sulfate binding protein. Expanding on these
findings, we are now using a syndecan knockout mouse line to explore the mechanism
of differential feeding in our designed mutants. Preliminary results indicate a reduction
in weight gain in knockouts compared to their wildtype littermates post peptide administration.
Collectively, these data show that the physiologically relevant form of AgRP, previously
considered unable to interact with syndecans, is indeed a heparan sulfate binding
protein. Furthermore, our designed mutants have differential affinities for GAGs,
with increased affinity correlating to increased feeding potency. Finally, as the
MC4R pathway is thought to be a viable target for wasting disorders such as cachexia,
we are interested in leveraging this data to improve the potency and stability of
our designed AgRP mutants. Taken together, this work aims to develop new insights
and probe the therapeutic potential of a critical metabolic pathway.
PE-013
Evidence of a proteolytic phenomenon in the starch binding domain of the α-amylase
from Lactobacillus amylovorus
Zaira Esmeralda Sánchez Cuapio1, Alejandra Hernández Santoyo2, Sergio Sánchez Esquivel1,
Romina Rodríguez Sanoja1
1Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México,
2Instituto de Química, Universidad Nacional Autónoma de México
α-amylases are glycoside-hydrolases that catalyze the hydrolysis of internal α-1,4
glycosidic bonds in starch and glycogen generating smaller oligosaccharides (1). These
multidomain proteins contain a catalytic barrel (β/α)8 and, in some cases, one or
more non-catalytic domains whose function is generally described as carbohydrate binding
module (CBM) and particularly as starch-binding domains (SBD). The SBD can bind granular
starch increasing the local concentration of substrate at the active site of the enzyme
and may also disrupt the structure of the starch surface (2). The α-amylase from Lactobacillus
amylovorus has a structure that consists of a catalytic domain (CD) and an unusual
carboxy-terminal starch-binding domain with 5 identical CBMs (belonging to family
26) in tandem (3). Each repeat acts as an independent fixing module with an additive
or synergic effect between the units (4). When we stored pure SBD from L. amylovorus
we found multiple forms of low molecular weight with a constant pattern, which does
not correspond to random degradation. Interestingly, when the protein is stored at
pH close to 5 and EDTA is added, such proteolysis appears to decrease. So far, there
is little information about the proteolytic process of amylases and the nature of
it. Here we show that divalent ions induce a proteolytic cleavage of the SBD, raising
the possibility of an autoproteolytic activity.
Acknowledgments:
This work is supported by grants PAPIIT IN222113-3 and CONACyT 131149. Sánchez Cuapio
Z is supported by a personal grant from Consejo Nacional de Ciencia y Tecnología,
México.
References:
1. Tibbot B., Wong D., Robertson G. & G. (2002). Biologia Bratislava. 11:229-238.
2. R. Rodríguez-Sanoja,* B. Ruiz, J. P. Guyot, and S. Sanchez (2005). Appl Environ
Microbiol. 71(1): 297–302.
3. Rodríguez Sanoja, R., Morlon-Guyot, J., Jore, J., Pintado, J., Juge, N. & Guyot,
J.P. (2000). Appl. Environ. Microbiol. 66: 3350-3356.
4. Guillén, D., Santiago, M., Linares, L., Pérez, R., Morlon, J., Sánchez, S. and
Rodríguez-Sanoja, R. (2007) Appl. Environ. Microbiol. 73:3833-3837.
PE-014
In Situ Membrane Protein Structure and Function Analysis using Site-Specific Unnatural
Amino Acid Incorporation and Spectroscopy Methods
Changlin Tian1
1University of Science and Technology of China
Unnatural amino acid and related methods provided a special mechanism to implement
site-specific spectroscopy active probe incorporation in a specific membrane protein
in cells. The site specific incorporation resulted in a single signal during acquisition,
resulting in unambiguous signal assignment. The protein specific labeling makes it
possible for in situ membrane protein analysis using NMR or fluorescence detection.
The 19F containing unnatural amino acid incorporation has been applied for dynamic
studies of transporters in native lipid membrane, and the phosphorylation quantification
analysis for Tyrosine kinase in native lipid membrane with the aid of lipodisc. The
fluorescent unnatural amino acid incorporation enabled the site-specific channel responses
analysis upon ligand binding in a single cell.
PE-015
Theoretical Volume Profiles as a Tool for Probing Protein Folding Kinetics
Heather Wiebe1, Noham Weinberg1,2
1Department of Chemistry, Simon Fraser University, 2Department of Chemistry, University
of the Fraser Valley
The mechanism by which conformational changes, particularly folding and unfolding,
occur in proteins and other biopolymers has been widely discussed in the literature.
Molecular dynamics (MD) simulations of protein folding present a formidable challenge
since these conformational changes occur on a time scale much longer than what can
be afforded at the current level of computational technology. Transition state (TS)
theory offers a more economic description of kinetic properties of a reaction system
by relating them to the properties of the TS, or for flexible systems, the TS ensemble
(TSE). The application of TS theory to protein folding is limited by ambiguity in
the definition of the TSE, although the experimentally observed first-order kinetics
for folding of small single-domain proteins lends itself to interpretation by this
theory. The pressure dependences of the folding rate constant can be used to obtain
activation energies and activation volumes, which are rationalized as the properties
of the folding TSE. The large amount of activation volume data in the literature has
gone largely uninterpreted at the quantitative level. We propose to utilize this data
in conjunction with MD-calculated volumetric properties to identify the TSE for protein
folding. The effect of pressure on reaction rates is expressed in terms of logarithmic
pressure derivatives, known as activation volumes. According to TS theory, activation
volumes can be identified as the difference in volume between the TS and reactant
species:
Activation volumes ΔV‡ have been experimentally determined for the folding of several
proteins. The concept of activation volume can be extended to that of a volume profile,
ΔV(y), which describes how the volume of a system changes along reaction coordinate
y. If the position y‡ of the TS along the reaction coordinate is unknown, it can be
found by locating ΔV‡ on the volume profile:
Such volume profiles can be built using our recently developed MD-based displacement
volume method.* Using this method, volumes of single molecules can be calculated by
taking the difference between the volume of pure solvent and solvent containing the
desired solute. This method takes into account the strength and type of solvent-solute
interactions as well as the geometrical configuration of the solute. In this work,
we present the successful application of this method to several conformationally flexible
systems.
*H. Wiebe, et al., J. Phys. Chem. C 116, 2240 (2012)
H. Wiebe and N. Weinberg, J. Chem. Phys. 140, 124105 (2014).
PE-016
Structure of the P15PAF/PCNA complex and implications for clamp sliding on the DNA
during replication and repair
Alfredo De Biasio1, Alain Ibáñez de Opakua1, Gulnahar Mortuza2, Rafael Molina3, Tiago
Cordeiro4, Francisco Castillo5, David Gil-Cartón1, Pau Bernadó4, Guillermo Montoya2,
Francisco Blanco1,6
1Structural Biology Unit, CIC bioGUNE, Derio, Spain, 2Novo Nordisk Foundation Center
for Protein Research, University of Copenhagen, 3Structural Biology and Biocomputing
Programme, CNIO, Madrid, Spain, 4Centre de Biochimie Structurale, Université Montpellier
1 and 2, France, 5Department of Physical Chemistry, Universidad de Granada, Spain,
6IKERBASQUE, Basque Foundation for Science
The intrinsically disordered protein p15PAF is overexpressed in cancer and regulates
DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA)
sliding clamp [1,2,3]. We have characterized the structure of the human p15PAF/PCNA
complex by NMR, crystallography, and computational modeling [4]. The central PCNA
interacting protein motif (PIP-box) of p15PAF is tightly bound to the canonical PIP-box
binding groove on the PCNA front face. In contrast to other PCNA interacting proteins,
however, p15PAF also contacts the inside of, and passes through, the PCNA ring. The
mostly disordered p15PAF chain termini thus emerge at opposite faces of the ring,
but remain protected from degradation by the 20S core proteasome. We also unveil a
novel DNA binding activity of p15PAF, both free and bound to PCNA, which is mainly
mediated by its conserved histone-like N-terminal tail. Molecular modeling shows that
a ternary complex with a duplex DNA inside the PCNA ring is energetically feasible
and our electron micrographs show increased density inside the ring. We propose that
p15PAF acts as a flexible drag that regulates PCNA sliding along the DNA, and may
facilitate the switch from replicative to translesion synthesis polymerase binding
upon DNA damage.
Acknowledgements:
This work has been mainly sponsored by MINECO grant CTQ2011-28680 and Juan de la Cierva-2010
contract to Alfredo De Biasio.
References:
[1] Alfredo De Biasio, et al. p15(PAF) Is an Intrinsically Disordered Protein with
Nonrandom Structural Preferences at Sites of Interaction with Other Proteins. Biophys
J 106, 4, 865-74, 2014.
[2] Chanlu Xie et al. Proliferating cell unclear antigen-associated factor (PAF15):
A novel oncogene. Int J Biochem Cell Biol 50, 127-31, 2014.
[3] Alfredo De Biasio and Francisco J. Blanco. Proliferating cell nuclear antigen
structure and interactions: too many partners for one dancer? Adv Protein Chem Struct
Biol 91, 1-36, 2013.
[4] Alfredo De Biasio et al. Structure of p15(PAF)-PCNA complex and implications for
clamp sliding during DNA replication and repair. Nat Commun. 12, 6, 6439, 2015.
PE-017
DHRS7 enzyme – important player in human health and diseases?
Lucie Zemanova1, Hana Stambergova1, Tereza Lundova1, Rudolf Andrys1, Jiri Vondrasek2,
Vladimir Wsol1
1Faculty Of Pharmacy in Hradec Kralove, Charles University in Prague, 2Institute of
Organic Chemistry and Biochemistry, AS CR
Metabolic syndrome (MetS) is one of the leading causes of the death worldwide; however,
exact pathophysiological mechanisms of MetS remain largely unknown. Growing evidence
suggests that the increased availability of glucocorticoids at the tissue level play
an important in MetS development. One of the major determinants of glucocorticoid
local action seems to be the enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1).
This enzyme is a well-known member of the short-chain dehydrogenase/reductase (SDR)
superfamily. It is an important carbonyl reducing enzyme that, besides its role fine-tuning
of glucocorticoids actions, is involved in the biotransformation of drugs and in the
development of lung cancer through metabolism of the tobacco specific carcinogen NNK.
The phylogenetically closest relative of 11β-HSD1 is DHRS7 enzyme from the same superfamily.
Unlike 11β-HSD1, DHRS7 is poorly characterized however it can be supposed at least
partially overlapping function to 11β-HSD1. Moreover its possible association with
similar pathological conditions in human as 11β-HSD1 has already been indicated by
several studies. The aim of this study is the basic biochemical characterization of
DHRS7. The enzyme is a member of cluster 3 of “classical” SDR; such members are considered
to be retinoid and steroid metabolizing enzymes, so characterization the enzyme was
based on this assumption. DHRS7 was prepared in recombinant form in the Sf9 cell line.
It was proved that this enzymes is an integral membrane-bound enzyme localized in
the endoplasmic reticulum with luminal orientation, similarly to 11β-HSD1. Known substrates
of 11β-HSD1 and related enzymes were tested also as substrates of DHRS7. It was proved
that DHRS7 is NADPH-dependent reductase with important substrates as steroid hormones
cortisone and androstene-3,14-dione, all-trans-retinal and also xenobiotics as 1,2-naphtoquinone
or carcinogen NNK at least in vitro. For better understanding of the catalytic function
of DHRS7 its structural model was prepared and it is used also for the identification
of additional substrates by ligand virtual screening. DHRS7 enzyme is expressed in
several human tissues as adrenals, liver, prostate, small intestine and kidney. These
brand new initial results point to the possible involvement of DHRS7 in important
cellular processes that deserve further investigation.
These results will lay the foundation for an understanding of DHRS7 role in human
physiology resp. pathophysiology.
This project was supported by Grant Agency of Charles University (677012/C/2012) and
UNCE 204026/2012).
PE-018
Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease
with both DNA nicking and Rnase activities
Bong-Jin Lee1, Ki-Young Lee1
1College of Pharmacy, Seoul National University
HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we
determined the solution structure of HP0268 using three-dimensional nuclear magnetic
resonance (NMR) spectroscopy, revealing that this protein is structurally most similar
to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity.
We also demonstrated for the first time that HP0268 is a nicking endonuclease and
a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy.
The nuclease activities for DNA and RNA were maximally increased by Mn(2+) and Mg(2+)
ions, respectively, and decreased by Cu(2+) ions. Using NMR chemical shift perturbations,
the metal and nucleotide binding sites of HP0268 were determined to be spatially divided
but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered
and form the nucleotide binding site. Moreover, site-directed mutagenesis was used
to define the catalytic active site of HP0268, revealing that this site contains two
acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding
and active sites are not conserved in the structural homologues of HP0268. This study
will contribute to improving our understanding of the structure and functionality
of a wide spectrum of nucleases.
PE-019
High-fidelity recombinant protein production in a silkworm bioreactor
Sungjo Park1, In-Wook Hwang1, Tatsuya Kato2, Enoch Park2, Andre Terzic1
1Center for Regenerative Medicine, Mayo Clinic, 2Laboratory of Biotechnology, Shizuoka
University
The domesticated silkworm, Bombyx mori, is an attractive host naturally equipped with
a proficient post-translational modification machinery adequate to fulfill stringent
demands of authentic recombinant protein production. Silkworm-based protein expression
has originally relied on a prototype baculovirus vector system that employs silkworm
as a bioreactor in place of more traditional cell lines. Recent development of the
silkworm trophic B. mori nucleopolyhedrovirus (BmNPV) bacmid launches a second generation
of silkworm-based protein production technology. Introducing the recombinant bacmid
DNA into silkworms expedites heterologous protein expression by eliminating prior
virus construction and amplification steps. Salient examples of heterologous eukaryotic
proteins produced in silkworms are acetyl-CoA carboxylase 2, malonyl-CoA decarboxylase,
Spot14/Mig12 heterodimer and α2,6-sialyltransferase with consistent high levels of
protein expression. Thus, equipped with a fail-safe post-translational modification
machinery, eukaryotic proteins are readily bioengineered using a silkworm-based protein
expression platform.
PE-020
Studies exploring potential applications of synthetic antifreeze proteins in the frozen
food industry
Ho Zee (Charles) Kong1, Conrad Perera1, Ivanhoe Leung1, Nazimah Hamid2, Viji Sarojini1
1School of Chemical Sciences, The University of Auckland., 2School of Applied Sciences,
Auckland University of Technology
In nature, certain species of plants, insects and fish produce a group of antifreeze
glycoproteins and polypeptides which enable them to survive the freezing temperatures
of their natural habitat. These naturally occurring antifreeze proteins (AFPs) were
first discovered in polar fishes such as antarctic notothenioids and winter flounder.
These AFPs have the ability to bind to ice crystals and restrict their size and morphology;
decrease the freezing point of water and inhibit the ice recrystallization processes.
Ice crystal formation is of primary concern to the frozen food industry, as ice crystal
formation during freezing can be disruptive to and cause damage to the cellular structures
in food. The unique properties of AFPs can be developed into a potential solution
to minimize freeze-thaw damage to frozen food. A number of tailor made synthetic analogues
based on the naturally occurring AFPs were successfully designed and synthesized.
Antifreeze activity studies of the AFPs were carried out using the Clifton nanoliter
osmometer attached with a microscope. The AFPs exhibited thermal hysteresis as well
as modification of ice crystal morphology, confirming their antifreeze activity in
vitro. The ability of these synthetic AFPs in preserving the texture and structure
of frozen food was evaluated using the techniques of scanning electron microscopy.
The AFPs showed great potential to preserve the cellular structures of frozen food
samples during freeze-thaw process. Additionally, secondary structure analysis of
the AFPs was carried out using circular dichroism. This presentation will summarize
our current results on the design, synthesis and anti-freeze activity analysis of
the synthetic AFPs.
PE-021
Development of Fungal-Specific Calcineurin Inhibitors Based on Molecular Structure
and Dynamics
Ronald Venters1, Leonard Spicer1,2, Joseph Heitman3, William Steinbach4, Praveen Juvvadi4,
Maria Schumacher2
1Duke University NMR Center, 2Duke University Biochemistry Department, 3Duke University
Department of Molecular Genetics and Microbiology, 4Duke University Department of
Pediatrics
Invasive fungal infections remain a leading cause of death in immunocompromised patients.
Current antifungal agents have a host of issues including limited efficacy, host toxicity
and an alarming increase in resistance. Current research in our laboratories is focused
on targeting the calcineurin signaling pathway that has been shown to be required
for fungal pathogenesis. Calcineurin is a highly conserved serine-threonine-specific
Ca2+-calmodulin-activated phosphatase important in mediating fungal pathogenesis and
stress responses. It is a key regulator of a signal transduction network required
for survival of the most common pathogenic fungi in humans, making it an ideal target
for fungal drug development. Calcineurin is a heterodimer of a catalytic (A) and regulatory
(B) subunit. Phosphatase activity requires association of the two subunits. Calcineurin
is also the target of the immunosuppressant FK506, which functions as an inhibitor
by first complexing with the peptidyl-prolyl cis-trans isomerase immunophilin, FKBP12.
The FKBP12-FK506 complex subsequently binds to calcineurin in a groove between the
A and B subunits and inhibits its activity. Although fungal calcineurins are targeted
by FK506, it also targets mammalian calcineurin and is thus immunosuppressive in the
host. In order to improve therapeutic efficacy, we have undertaken a unique effort
that utilizes both structural biology and molecular mycology in an effort to overcome
the fungal versus human specificity barrier. The NMR studies to be presented here
have been focused on determining the resonance assignments and solution structures
for the FKBP12 proteins from the pathogenic fungi Candida albicans, Candida glabrata
and Aspergillus fumigatus. Notably, the X-ray crystallography structures of the wild-type
Candida albicans and Aspergillus fumigatus FKBP12 proteins revealed an intriguing
intermolecular interaction involving four residues in the 80’s loop including Pro104
(in C. albicans) and Pro90 (in A. fumigatus) which are stabilized in the cis conformation.
These data suggest that the protein might use itself as an enzyme substrate. In efforts
to establish if this interaction remains in a solution environment, we have determined
the NMR structure and measured the T2 relaxation rates for the wild-type A. fumigatus
FKBP12 protein and for the P90G mutant variant that adopts a dramatically different
orientation of the 80’s loop and does not form an intermolecular interaction in the
crystal structure. The NMR chemical shift data indicate that, while the remainder
of the protein structure remains unchanged, the 80’s loops in the two variants are
indeed different. In addition, the T2 relaxation rates of the residues in this region
are dramatically dissimilar in the two variants, but remain identical throughout the
rest of the protein. We have also begun inhibitor binding studies of all of the FKBP12
proteins from each of the pathogens by titrating the FK506 inhibitor into native and
mutant FKBP12 proteins in order to examine conformational changes associated in the
protein upon complex formation. Using this approach we plan to determine the relative
Kd values for binding of each inhibitor to the FKBP12 protein from each pathogen for
comparison of binding proclivities.
PE-022
Lupin (Lupinus angustifolius L.) b-conglutin proteins: Structure functional features,
catalytic mechanism modeling and cross-allergenicity identification using protein
threading and molecular docking methods
Jose C. Jimenez-Lopez1,2
1Department of Biochemistry, Cell and Molecular Biology of Plants; EEZ-CSIC, 2The
UWA Institute of Agriculture; The University of Western Australia
Lupin is an important PULSE, which displays a wide range of benefits in agriculture,
particularly these involved in possible plant pathogen suppression. Furthermore, lupin
seed proteins promote different positive health aspects, preventing cardiovascular
disease, and reduction of glucose and cholesterol blood levels. “Sweet lupine” seeds
seem to be promising as a source of innovative food ingredients due to averaged protein
content similar to soybean and an adequate composition of essential amino acids. Thus,
lupin seeds may be important source of proteins for human and animal consumption.
However, and as drawback feature, the number of allergic people to lupin seed proteins
is rising, becoming a serious and a growing problem in the Western world, because
of the rapid introduction of lupin seeds as new ingredients in traditional and novel
foods. The goals of this study are the characterization the structure-functional properties
of Lupinus angustifolius L or Narrow leafed lupin (NLL) β-conglutin proteins, with
a focus in its catalytic mechanism, and its molecular cross-allergenicity with other
legumes, i.e. peanut, by extensive analysis using different computer-aided molecular
approaches covering (i) physicochemical properties and functional-regulatory motifs,
(ii) sequence analysis, 2-D and 3D structural (threading) modeling comparative study
and molecular docking, (iii) conservational and evolutionary analysis, (iv) catalytic
mechanism modeling, and (v) sequence, structure-docking based β-cell epitopes prediction,
while T-cell epitopes were predicted by inhibitory concentration and binding score
methods. β-conglutins (vicilin-like or 7S proteins) are seed proteins typically found
in reserve tissues (endosperm and cotyledon). They belong to the Cupin superfamily
of proteins, containing a globular domain constituted by a conserved b-barrel. Two
barrels were found in all β-conglutin protein isoforms and an additional mobile N-terminal
arm constituted bye α-helices. Molecular modeling analysis has shown that one of this
barrel contain a semi-conserved metal binding motive (HYX…R), typically found in Oxalate
oxidase (OXOX) enzymes. Interestingly, our results revealed considerable structural
differences between β-conglutin isoforms, particularly affecting 2-D elements (loops
and coils), and numerous micro-heterogeneities are present in fundamental residues
directly involved in epitopes variability, which might be a major contributor to the
observed differences in cross-reactivity among legumes. We also identified multiple
forms of β-conglutins polypeptides ranging from 15-80kDa, with IgE-binding characteristics
in atopic patients. Thus, β-conglutins might be considered as major allergen in different
species of lupin, including the “sweet lupin” group, since several of these polypeptides
were recognized by human IgEs, having the potential to trigger an immune response
leading to allergy symptoms.
Acknowledgements:
This research was funded by the European Research Program MARIE CURIE (FP7-PEOPLE-2011-IOF)
under the grant ref. number PIOF-GA-2011-301550.
PE-023
Intracellular pH and quinary structure
Rachel Cohen1, Gary Pielak1,2,3
1Department of Chemistry, University of North Carolina, 2Department of Biochemistry
and Biophysics, University of North Carolina, 3Lineberger Comprehensive Cancer Center,
University of North Carolina
NMR spectroscopy can provide information about proteins in living cells. pH is an
important characteristic of the intracellular environment because it modulates key
protein properties such as net charge and stability. Here, we show that pH modulates
quinary interactions, the weak, ubiquitous interactions between proteins and other
cellular macromolecules. We used the K10H variant of the B domain of protein G (GB1,
6.2 kDa) as a pH reporter in Escherichia coli cells. By controlling the intracellular
pH, we show that quinary interactions influence the quality of in-cell 15N-1H HSQC
NMR spectra. At low pH, the quality is degraded because of the increased number of
attractive interactions between E. coli proteins and GB1, which slows GB1 tumbling
and broadens its crosspeaks. Our results demonstrate the importance of quinary interactions
for furthering our understanding of protein chemistry in living cells.
PE-024
Advanced analytical tools for monitoring and control in production processes of recombinant
hemagglutinin influenza vaccine
Joanna Szewczak1, Weronika Surmacz-Chwedoruk1, Bożena Tejchman-Małecka1, Jacek Stadnik1,
Grażyna Tronczyńska-Lubowicz1, Agnieszka Romanik-Chruścielewska1, Jarosław Antosik1,
Piotr Borowicz1, Iwona Sokołowska1, Dorota Stadnik1,
1Institute of Biotechnnology and Antibiotics
Influenza virus is one of the most prevalent pathogens causing respiratory illness
which often leads to serious post influenza complications such as pneumonia and myocarditis.
Some viruses, as the avian influenza H5N1, are especially dangerous and draw special
attention of WHO. This highly pathogenic virus spreads quickly among domestic poultry
and wild birds resulting in high mortality. What is more distressing, the H5N1 virus
may be transmitted to humans. Because of antigenic drift it is impossible to deliver
an effective vaccine against all subtypes of the H5N1 virus. Moreover, traditional
egg-based production of influenza vaccines is time- and cost-consuming, what makes
it inadequate in case of a pandemic. Hence, we have developed an efficient production
process of influenza vaccine based on a recombinant hemagglutinin antigen (rHA). Recombinant
vaccines underlay strict regulations and quality requirements. The purpose of this
work was to develop a battery of analytical methods that allow to evaluate key quality
attributes of rHA on each stage of production. At first, we have focused on rHA structure
as a crucial issue for its activity. The primary structure of rHA was confirmed by
peptide mapping and TOF/TOF fragmentation (HPLC, MALDI TOF/TOF). Furthermore, FTIR
analysis was used to evaluate the secondary structure of the protein. The disulfide
bonds, which stabilize the tertiary structure, were assigned by peptide mapping. Additionally,
free thiols were measured using Ellman’s reagent. Moreover, we have employed RP-HPLC,
SEC-MALS and DLS to explore oligomerization of rHA. These techniques appeared to be
useful not only to confirm existence of native oligomers, but also to find and discard
misfolded fraction, aggregates and truncated forms. In addition, two analytical methods
(RP-HPLC and CGE) were developed to assess the purity of rHA as required by ICH guidelines.
We also have determined isoelectric point and heterogeneity of rHA by cIEF. Afterward,
developed methods were applied in the stability studies that provide a valuable insight
into a chemical degradation process and conformational changes of rHA during storage.
This work was supported by Innovative Economy Operational Program, Grant No. WND-POIG.01.01.02-00-007/08-00
as a part of project “Centre of medicinal product biotechnology. Package of innovative
biopharmaceuticals for human and animal therapy and prophylactics.”
PE-025
Muscle cell atrophy via HSP gene silencing was counteracted by celastrol-mediated
HSP overexpression
Inho Choi1, Taesik Gwag1, Kyoungsook Park2, Kyoungbong Ha1, Joo-hee Lee3, Youn-Kyu
Kim3
1College of Science and Technology, Yonsei University, 2Molecular Therapy Research
Center, Sungkyunkwan University, 3Korea Aerospace Research Institute
Molecular chaperone heat shock proteins (HSP) are known to assist protein quality
control under various stresses. Although overexpression of HSP70 was found to promote
muscle mass retention in an unloading state, it is unclear whether muscle atrophy
is induced by suppression of HSP expression and is counteracted by active HSP overexpression.
In this study, we pre-treated Hsp70 siRNA to rat L6 cells for the HSP gene-silencing,
and determined myotube diameter, HSP72 expression and anabolic and catabolic signaling
activities in the absence or presence of triterpene celastrol (CEL), the HSP70 inducer.
Relative to a negative control (NC), muscle cell diameter was reduced by 11% in the
siRNA-treated group, increased 1.2-fold in the CEL-treated group and remained at the
size of NC in the siRNA+CEL group. HSP72 expression was decreased 65% by siRNA whereas
the level was increased 6- to 8-fold in the CEL and siRNA+CEL groups. Expression of
FoxO3 and atrogin-1 was increased 1.8- to 4.8-fold by siRNA, which was abolished by
CEL treatment. Finally, phosphorylation of Akt1, S6K and ERK1/2 was not affected by
siRNA, but was elevated 2- to 6-fold in the CEL and siRNA+CEL groups. These results
suggest that HSP downregulation by Hsp gene-silencing led to muscle cell atrophy principally
via elevation of catabolic activities. Such anti-atrophic effect was counteracted
by CEL-mediated HSP overexpression.
This abstract is sponsored by Executive Director Jody McGinness (jmcginness@proteinsociety.org)
PE-026
A novel in vivo characterization method predicting the physicochemical parameters
of an antibiotic efflux pump
Anisha M Perez1, Erin L O’Brien1, Marcella M Gomez1, Matthew R Bennett1, Yousif Shamoo1
1Department of BioSciences
The Centers for Disease Control and Prevention report that at least 2 million people
in the United States will become ill due to antibiotic resistant pathogens leading
to 23,000 deaths each year. In order to circumvent these resistance mechanisms, it
is essential to quantitatively understand how the function of the protein(s) involved
relates directly to resistance. Integral membrane efflux pumps are known determinants
of single-drug and multi-drug resistance in a wide variety of pathogenic organisms.
These transporters are proteins whose characterization typically requires reconstitution
in an artificial membrane. Subsequently, these important proteins are difficult to
characterize by traditional in vitro studies. My project aims to determine the physicochemical
parameters of the efflux pump TetB utilizing molecular biology and mathematical modeling.
TetB is composed of 12 transmembrane (TM) alpha-helices and is found within the inner
membrane of Gram-negative bacteria. This protein allows for the efflux of tetracycline
(TET), doxycycline (DOX), and minocycline (MCN) antibiotics from the cytoplasm into
the periplasm. These tetracyclines are a bacteriostatic class of antibiotics that
inhibit protein synthesis by binding to the 30S ribosomal, therefore, blocking the
binding of aminoacyl-tRNA. For cells grown in tetracyclines, the efflux mechanism
of TetB decreases the cytosolic antibiotic concentration allowing for the rate of
protein translation to increase. I have inserted a tet(B) expression system into the
chromosome of an Escherichia coli lab strain and have determined its growth profile
under various concentrations of TET, MCN, and DOX using a high-throughput 96-well
plate format. The growth rate profiles correlate with TetB pumping rates for each
drug. TetB more readily pumps out TET compared with DOX and MCN and we observe that
cells expressing TetB can grow at higher TET concentrations compared with DOX and
MCN. The shapes of the growth rate profiles produced in the different drugs give insight
into the physicochemical mechanism of TetB. We have built a preliminary mathematical
model that can simulate these growth profiles and predict efflux pump physicochemical
parameters. We are currently working on understanding how efflux expression effects
bacterial growth by testing ribosome binding site (RBS) sequences of varying strengths
in our tet(B) expression system. Future work is geared toward modeling more complex
efflux pumps such as the tripartite pumps which traverse both bacterial membranes
and cause multi-drug resistance. Collectively, this project aims to build an in vivo
system which will allow for the characterization of a variety of efflux pumps without
the arduous tasks of protein purification and subsequent reconstitution.
PE-027
Structural Analysis of KCNE1 Transmembrane Mutant Yielding KCNE3-like Function
Cheryl Law, Charles Sanders
1Vanderbilt University Biochemistry Department, 2Vanderbilt University Center for
Structural Biology, 3Vanderbilt University School of Medicine
The KCNE family contains five single transmembrane-spanning proteins that modulate
several channels including the voltage-gated potassium channel, KCNQ1. These five
proteins provide functional diversity to the KCNQ1 channel. For example, the KCNQ1/KCNE1
complex leads to a slow and delayed opening of the channel while the KCNQ1/KCNE3 complex
leads to a rapid opening and constitutively active channel. Mutations in both the
KCNQ1 channel and members of the KCNE family have been shown to lead to disease such
as long QT syndrome, cardiac arrhythmias, deafness, and sudden death. Melman YF, et
al (2007) identified a small transmembrane region of both KCNE1 and KCNE3 that are
essential for their unique modulation of the KCNQ1 channel. By swapping a triplet
motif in the transmembrane region of KCNE1 and KCNE3, we can flip the primary function
of these two proteins. While the key for KCNE1 and KCNE3’s unique modulating is believed
to lie in this triplet motif, the mechanism and structural changes involved in this
modulation is not fully understood. By using NMR spectroscopy, biochemical studies,
and computational docking, we aim to look at the structural and conformational differences
between KCNE1 and the triple mutant KCNE1 substituted with the three essential KCNE3
residues. We have expressed and purified 15N-labled KCNE1 triple-mutant in sufficient
quantities for NMR studies in LMPG detergent micelles and other membrane mimetics,
and we have collected 2D NMR spectra using a TROSY-based pulse sequence. Partial backbone
assignments of KCNE1 triple mutant have been determined by aligning and transfer assignments
of the WT KCNE1 previous determined in our lab. With the structure of KCNE1 triple
mutant determined, we aim to computationally dock the triple mutant into a model of
the full-length KCNQ1 channel in the open and closed state. Lastly, we will compare
the known structure of KCNE1 docked to a model of KCNQ1 to that of the KCNE1 triple
mutant to determine key interactions, significant structural and conformational changes,
and how the triple motif region gives rise to its specific structural and functional
differences. With this information, we can begin to understand the mechanism of the
functional diversity of the KCNE family on KCNQ1 potassium channel.
PE-028
Biochemical characterization of Brassica napus diacylglycerol acyltransferase 1 and
its regulatory domain
Kristian Mark Caldo1, Rashmi Panigrahi2, Michael Greer1, Guanqun Chen1, M. Joanne
Lemieux2, Randall Weselake1
1Alberta Innovates Phytola Centre, University of Alberta, 2Department of Biochemistry,
University of Alberta
Diacylglycerol acyltransferase 1 (DGAT1) is a membrane-bound enzyme catalyzing the
final and committed step in the acyl-CoA-dependent triacylglycerol (TAG) biosynthesis.
The level of DGAT activity in Brassica napus seeds has a substantial effect on the
flux of carbon towards TAG formation. Although membrane-bound DGATs have been investigated
for a long time, their modes of action and regulation remain poorly understood due
to difficulties encountered during purification. This study aimed to purify and characterize
recombinant Brassica napus DGAT1 to increase our understanding of the regulation of
seed oil formation. Herein, we describe the purification of active recombinant B.
napus DGAT1 (BnaC.DGAT1.a) expressed in Saccharomyces cerevisiae. Purified BnaDGAT1
in n-dodecyl-β-D-maltopyranoside (DDM) micelles behaves as dimers, which can associate
further to form tetramers. The acyl donor preference of the major dimeric form with
sn-1,2-diolein as acceptor follows the following order: α-linolenoyl-CoA > oleoyl-CoA = palmitoyl-CoA > linoleoyl-CoA > stearoyl-CoA.
The first 113 residues of BnaC.DGAT1.a corresponding to a soluble regulatory region
was expressed in Escherichia coli and purified. Truncation of this soluble domain
reveals that the dimeric interface is located within residues 49-113, while the first
48 residues allow formation of tetramers. This N-terminal region was implicated as
an allosteric exosite for acyl-CoAs as revealed by previous Lipidex-1000 binding studies.
In the current study, circular dichroism spectroscopy and isothermal titration calorimetry
were used to probe the binding kinetics and thermodynamics. DGAT1 appears to shift
between two oligomerization states, a phenomenon that may be related to regulation
of enzyme activity and mediated by the N-terminal domain.
PE-029
Alteration of lysine and arginine content as a strategy to modify protein solubility:
a test for E. coli proteins
M. Alejandro Carballo-Amador1, Jim Warwicker1, Alan J. Dickson1
1Faculty of Life Sciences, University of Manchester
Protein aggregation is an undesired physicochemical mechanism whether for biophysical
and structural studies or for biopharmaceutical companies, at any scale. In Escherichia
coli, protein accumulation in the cytoplasm can result in protein aggregation to form
what are known as inclusion bodies (IBs). Several experimental approaches have been
undertaken to prevent protein aggregation. However, there is no universal approach
or technology that solves protein aggregation. Recently, our group found that the
sequence-based property of lysine versus arginine content separated E. coli proteins
by solubility. In this study, we investigated solubility alterations for three highly
soluble E. coli proteins (thioredoxin-1 [TRX], cold shock-like protein cspB [cspB],
and the histidine-containing phosphocarrier protein [HPr]), with varying degree of
lysine substitution by arginine. These experiments are predicted to decrease the solubility
of the variants, according to our computational calculations. Our findings revealed
a significant decrease in solubility for cspB and HPr, which is more evident in variants
with low or null lysine content. However, for the expression of TRX variants, solubility
only falls under low induction conditions (low temperature and IPTG inducer) compared
to WT. This computational and experimental approach is a first step in studying to
what extent the lysine:arginine ratio modifies solubility.
PF-001
Role of C terminal disordered domain of Sesbania mosaic virus RNA dependent RNA polymerase
in the modulation of its activity and oligomeric status
Arindam Bakshi1, Srinivas Sistala2, Shruthi Sridhar1, Savithri H S1
1Indian institute of Science, 2Wipro G E Healthcare Pvt Ltd
Sesbania mosaic virus (SeMV) is a single stranded positive sense RNA virus with a
genome length of 4148 nucleotides and belongs to the genus sobemoviruses. SeMV RNA
dependent RNA polymerase (RdRp) (62 KDa) was previously shown to interact strongly
with virus encoded P10 protein (10 KDa). Such an interaction was found to increase
the activity of RdRp in vitro. Further, deletion of C terminal 43 amino acid residues
also resulted in increase in the polymerase activity that was comparable to the full
length RdRp-P10 complex. It was proposed that the conserved C terminal disordered
domain of RdRp was responsible for interaction with P10 and modulation of the activity.
In the present study, role of the C terminal disordered domain was further investigated
by determining the oligomeric status of the complex and the C terminal deletion mutants
of RdRp and also by quantitating the RdRp-P10 interaction using surface plasmon resonance.
Size exclusion chromatography revealed that RdRp eluted in the void volume of the
column whereas a significant fraction of the RdRp-P10 complex eluted at a position
corresponding to the size of the 1:1 complex of RdRp and P10 (77KDa). Activity measurements
indicated that the heterodimeric complex was more active than the aggregate eluting
in the void fraction. Interestingly, the C terminal deletion mutants of RdRp (C del
43 & C del 72 RdRp) were also found to be less aggregated as compared to full length
RdRp and some of the protein eluted at a position corresponding to the respective
monomers. These monomers were also more active than the aggregate fractions. These
results demonstrate that the increase in activity observed either upon interaction
with P10 or deletion of the C terminal domain could be due to the change in the oligomeric
state of RdRp. In order to further analyze the interaction of RdRp with P10 surface
plasmon resonance was used. RdRp and its deletion mutants were immobilized on Biacore
sensor surface and P10 protein was used as an analyte. Full length RdRp and C del
43 RdRp were shown to interact with P10 with KD values of 0.6 and 1 uM respectively.
However, C del 72 and C del 85 RdRp did not show any binding with P10. These results
suggest that the region 43-72 from the C terminus of RdRp is essential for the interaction
with P10. Further, the C del 85 RdRp was inactive although C del 72 RdRp continued
to be active suggesting that residues 72-85 from the C terminus are crucial for RdRp
activity. Further studies are in progress to identify the residues within these motifs
that may be essential for the activity or interaction with P10.
PF-002
Aggregation of androgen receptor in spinal bulbar muscular atrophy is a multistep
process
Giulio Chiesa1, Bahareh Eftekharzadeh1, Daniele Mungianu1,2, Alessandro Piai2, Jesus
Garcia1, Isabella Felli2, Roberta Pieratelli2, Xavier Salvatella1,3
1Institute for Research in Biomedicine (IRB), 2Magnetic Resonance Center and Department
of Chemistry, University of Florence, 3ICREA
Spinal bulbar muscular atrophy (SBMA) is a member of the polyglutamine (polyQ) expansion
diseases, like Huntington disease, and it is caused by a genetic expansion of the
polyCAG tract in exon 1 of androgen receptor (AR) that codes for the polyQ region.
SBMA is a late onset disease, which involves a progressive degeneration of the motor
neurons and consequent muscular atrophy. There is still no treatment available for
this disease. AR is a nuclear receptor that responds to testosterone and that regulates
the expression of the masculine phenotype. It is composed of an intrinsically disordered
N-terminal domain (NTD) that bears the polyQ tract, a DNA binding domain and a ligand
binding domain. Aggregates of AR protein with an extended polyQ are observed in the
motor neurons of SBMA patients. In vitro studies showed that aggregation of Androgen
Receptor takes place only in presence of testosterone1 and that the cleavage of the
protein by caspase 3 is a crucial event for cytotoxicity. However, there is no clear
knowledge of the mechanism of aggregation, for this protein. An increasing body of
evidence supports the hypothesis that the aggregation of these proteins is controlled
by regions flanking the polyQ tract, by regulating the rate of aggregation depending
on their secondary structure. We have applied nuclear magnetic resonance (NMR) and
circular dichroism for generating information on the secondary structure of the N-terminal
cleavage product of AR by caspase 3 and we have studied its aggregation with a set
of biophysical methods, like dynamic light scattering, an HPLC sedimentation assay
and transmission electron microscopy. We have found that the polyQ tract of AR presents
a high degree of helicity. We attribute this conformation to the N-terminal flanking
region, characterized by high helicity and we have tested this hypothesis by performing
mutations. We have also observed that the rate of the first step of oligomerization
is not dependent on the number of glutamine repeats, but instead is due to self interactions
of a region N-terminal to and far from the polyQ. Its progression to fibril is dependent
to the number of glutamines in the tract. We have therefore identified two steps in
the aggregation process of AR, where a motif far from the polyQ at its N-terminal
drives the early oligomerization, followed by the interaction of the polyQ chains
that stabilize it and determine the progression to fibrils. These findings shed a
light for possible interventions on the AR oligomerization process, thus suggesting
a different strategy to study the onset of the disease in SBMA patients.
PF-003
Destabilizing the Transient Helical Conformation of Islet Amyloid Polypeptide Hastens
Peptide Self-Assembly and Potentiates Cytotoxicity
Carole Anne de Carufel1, Phuong Trang Nguyen1, Alexandre Arnold1, Isabelle Marcotte1,
Steve Bourgault1
1University of Quebec in Montreal, Department of Chemistry
Amyloidogenic polypeptides can be divided into two different structural classes: those
that are intrinsically disordered and those that show a well-defined structure in
their monomeric soluble state. Natively folded proteins, such as transthyretin, have
to unfold (or misfold), at least partially, to form amyloids. In contrast, intrinsically
disordered polypeptides, such as the islet amyloid polypeptides (IAPP) and Abeta peptide,
need to undergo conformational rearrangements allowing the formation of locally ordered
structure(s) to initiate the amyloidogenic process. Studies have shown that IAPP and
Abeta adopt an alpha-helix conformation in the initial steps of amyloidogenesis. This
intermediate is believed to be on-pathway to fibril formation, although this hypothesis
is still the matter of debate. In this study, we designed human IAPP (hIAPP) derivatives
in which alpha-helix destabilizing substitutions were incorporated into the putative
helical segment of IAPP to probe the initial structural event in amyloid formation.
Using trifluoroethanol titration, we observed by CD spectroscopy that strategic incorporation
of D-amino acids at positions 15 and 16 leads to an IAPP derivative (dIAPP) that cannot
fold into a helix. In homogeneous solution, hIAPP and dIAPP show similar kinetics
of fibrillization, as measured by Thioflavin T fluorescence. Although their amyloid
fibrils display different characteristics by AFM, IAPP and dIAPP are able to self-associate
to form amyloids when mixed together and when seeded with one another. Studies in
heterogeneous environment, notably in presence of glycosaminoglycans and model membranes
of DOPC/DOPG (7:3), showed a helical intermediate for hIAPP while only a beta-sheet
secondary structure was apparent for dIAPP. While the rate of amyloid fibril formation
was increased for both peptides, dIAPP was drastically affected by these anionic biomolecules
with an absence of lag phase. The incapacity of adopting a transient helical conformation
accentuates cell toxicity, supported by the caspase 3/7 activation level and the increase
in intracellular calcium level. Overall, this study indicates that the helical intermediate
is off-pathway to IAPP amyloid formation and offers novel mechanistic insights for
the development of molecular identities modulating peptide self-assembly and IAPP-induced
cytotoxicity.
PF-004
Towards in vivo NMR: Putting prions in context
Kendra Frederick1, Robert Griffin2, Susan Lindquist1,3,
1Whitehead Institute for Biomedical Research, 2Francis Bitter Magnet Lab and Department
of Chemistry, MIT, 3Howard Hughes Medical Institute, Department of Biology, MIT
For an organism to survive, its proteins must adopt complex conformations in a challenging
environment where macromolecular crowding can derail even robust biological pathways.
The situation is perilous: many diseases arise from improper folding of just a single
protein. To cope, cells employ a repertoire of molecular chaperones and remodeling
factors that usher unfolded proteins into active conformations, sequester them, or
target them for degradation. Yet, not all aggregated proteins are the result of mis-folding.
Yeast prions are self-templating protein-based mechanisms of inheritance that rely
upon chaperones for their propagation. The best studied of these is the prion domain
(NM) of Sup35, which forms an amyloid that can adopt several distinct conformations
(strains) that produce distinct phenotypes. Using genetic, biochemical, spectroscopic,
and solid state NMR techniques, we investigated the structural and dynamic underpinnings
of Sup35 amyloids and found that prion strains differ in both their atomic structure
as well as their dynamic motions. Interestingly, these mobility differences correlate
with differences in the interaction with molecular chaperones in vivo. Limitations
on the specificity and sensitivity of biophysical techniques typically restrict structural
investigations to purified systems at concentrations that are orders of magnitude
above endogenous levels. Therefore, I developed an approach to apply a sensitivity-enhancement
technique for NMR, dynamic nuclear polarization (DNP), to investigate interactions
between Sup35 and molecular chaperones at endogenous concentrations in their native
environments. Critically, I found that the cellular environment induced structural
changes in a region of Sup35 that is intrinsically disordered in purified samples
but known genetically to influence prion propagation from one generation to the next.
This approach enables structural and mechanistic investigation of proteins in biologically
relevant contexts.
PF-005
Genetic instability within regions encoding repetitive proteins as a driver of adaptation
Stephen Fuchs1
1Tufts University
More than ten percent of all eukaryotic proteins contain within them a region of repetitive
amino acid sequence. These repetitive domains range from short stretches of a single
amino acid to multiple copies of longer, heterogeneous amino acid sequences and generally
show lack of defined structure. They play diverse roles in cells including acting
as structural proteins, promoting cell-cell interactions, and mediating the assembly
of molecular machines. Tandem repeat proteins are known to be variable in length within
cellular populations although the mechanisms dictating this variability have not been
elucidated. Here we describe work uncovering specific features within the coding sequences
of repetitive proteins that contribute to tandem repeat instability in yeast. Furthermore,
we demonstrate that cells will expand and/or contract repetitive regions in order
to adapt to environmental stresses and describe a role for DNA repair proteins in
this process. Lastly, we demonstrate how these mechanisms are likely conserved in
higher eukaryotes, including humans. This study uncovers the molecular basis for an
important aspect of natural protein evolution and describes a novel mechanism for
adaptation in response to environmental changes.
PF-006
A Proline-Tryptophan turn in the intrinsically disordered domain 2 of NS5A protein
is essential for Hepatitis C virus RNA replication
Marie Dujardin1, Vanesa Madan2, Roland Montserret3, Puneet Ahuja1, Isabelle Huvent1,
Helene Launay1, Ralf Bartenschlager2, François Penin3, Guy Lippens1, Xavier Hanoulle1
1CNRS UMR 8576, UGSF, Lille University, 2Department of Infectious Diseases, Molecular
Virology, University of Heidelberg, 3CNRS UMR 5086, IBCP, LabEx Ecofect, Lyon 1 University
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the
human chaperone cyclophilin A (CypA), a peptidyl-prolyl cis-trans isomerase (PPIase),
are both targets for highly potent and promising antiviral drugs that are in late
stage of clinical development [1,2]. Despite its high interest in the development
of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional
protein poorly characterized at the molecular level. NS5A is required for HCV RNA
replication and is involved in viral particles formation and regulation of host pathways.
Thus far, no enzymatic activity or precise molecular function has been ascribed to
NS5A that is composed of a highly structured domain 1 (-D1), as well as two intrinsically
disordered domains 2 (-D2) and 3 (-D3). NS5A-D1 structure has been solved by X-ray
crystallography and NS5A-D2 and -D3 have been characterized by NMR spectroscopy. These
two last domains do not adopt a stable 3D structure but rather exist as an ensemble
of highly dynamic conformers. Using NMR spectroscopy, HCV NS5A-D2 has been shown to
establish a direct interaction with the human CypA and to be a substrate for the enzymatic
PPIase activity of CypA [3]. The CypA interaction site in NS5A-D2 is composed of nearly
15 residues that correspond to the most conserved region of the domain, with 3 Proline
residues being strictly conserved among all HCV genotypes. Whereas NS5A-D2 is mainly
disordered, some of its NMR resonances, corresponding to residues in the CypA binding
site, display unexpected 1H and 15N NMR chemical shifts for an intrinsically disordered
domain. Thus we have further characterized this region by NMR spectroscopy. A short
structural motif in the disordered NS5A-D2 has been identified and we solved its NMR
structure. In a cellular assay, we showed that this structural motif, a minimal Pro314-Trp316
turn, is essential for HCV RNA replication. We demonstrated that this Pro-Trp (PW)
turn is required for proper interaction with the host CypA and influenced its enzymatic
PPIase activity on residue P314 of NS5A-D2. This work provides a molecular basis for
further understanding of the function of the intrinsically disordered domain 2 of
HCV NS5A protein. In addition, our work highlights how very small structural motifs
present in intrinsically disordered proteins can exert a specific function.
[1]. Bartenschlager, R., Lohmann, V. & Penin, F. Nat. Rev. Microbiol. 11, 482–496
(2013).
[2]. Scheel, T. K. H. & Rice, C. M. Nat. Med. 19, 837–849 (2013).
[3]. Hanoulle, X. et al. J. Biol. Chem. 284, 13589–601 (2009).
PF-007
Solution structure and celullar functional studies of bovine cathelicidin Bt-6 (BMAP-27)
Anna Hastings1, Manuel Ruether2, H. Paul Voorheis1, Ken H. Mok1,3
1Trinity College Dublin, School of Biochemistry and Immunology, 2Trinity College Dublin,
School of Chemistry, 3TCD, Centre for Research on Adaptive Nanostructures and Nanodevices
(CRANN)
Solution structure and celullar functional studies of bovine cathelicidin Bt-6 (BMAP-27)
Anna Hastings1, Manuel Ruether2, H. Paul Voorheis1 and K. H. Mok1,3 1 Trinity College
Dublin, Trinity Biomedical Sciences Institute (TBSI), School of Biochemistry and Immunology,
Dublin 2, Ireland 2 Trinity College Dublin, School of Chemistry, Dublin 2, Ireland
3 Trinity College Dublin, Centre for Research on Adaptive Nanostructures and Nanodevices
(CRANN), Dublin 2, IrelandTrypanosoma brucei, the causative agent of African sleeping
sickness in humans and nagana in cattle, has been shown to be killed by a bovine antimicrobial
peptide, Bt-6 (BMAP-27) [1-2]. This 27-residue peptide also shows toxicity towards
mammalian cells but at higher concentrations, suggesting its possible usefulness as
a treatment for trypanosomiasis. Here we present the peptide’s relative cytotoxicity
for bloodstream and procyclic forms of T. brucei and for mammalian cells, the fate
of the peptide in T. brucei using fluorescently-labelled Bt-6, and its three dimensional
structure using NMR spectroscopy.Minimum inhibitory assays confirmed the peptide’s
selective toxicity towards both bloodstream and procyclic forms of T. brucei, demonstrating
its potential to serve as a starting point for a trypanocidal drug. Fluorescence spectrophotometric
experiments, carried out using fluorescein labelled Bt-6, show that the peptide is
released from the external surface of the parasite into the suspending medium under
de-energized conditions but retained in energized cells. Heteronuclear and homonuclear
biomolecular NMR experiments (TOCSY, NOESY, 1H-13C-HSQC,1H-15N-HSQC, etc) folowed
by structural calculations (chemical-shift based as well as simulated annealing techniques)
in the free state indicate that this peptide is mostly unstructured in aqueous solution,
suggesting that there is a major conformational change upon binding to T. brucei that
is required for uptake. We suggest that the evolutionary pressure that selected for
the intrinsically disordered structure of this peptide was the advantage it conferred
upon the host to bind to many different surface structures throughout the microbiological
world.
References:
[1] Haines, L. R. et al. (2003) Vector Borne Zoonotic Dis. 3: 175-186.
[2] Haines, L. R. et al. (2009) PLoS Negl. Trop. Dis. 3: e373.
PF-008
Engineered binding proteins to amyloidogenic intrinsically disordered proteins
Hamed Shaykhalishahi1,2, Ewa Mirecka1, Aziz Gauhar1, Clara Grüning1, Michael Wördehoff1,
Sophie Feuerstein2, Matthias Stoldt1,2, Torleif Härd3, Dieter Willbold1,2, Wolfgang
Hoyer1,2
1Physikalische Biologie, Heinrich Heine University, 2Structural Biochemistry (ICS-6),
Research Centre Jülich, 3Chemistry and Biotechnology, Swedish University of Agricultural
Sciences (SLU)
The misfolding and amyloid formation of proteins featuring intrinsically disordered
regions is a pathological hallmark of several neurodegenerative diseases, including
Alzheimer’s disease and Parkinson’s disease. Engineered binding proteins targeting
amyloidogenic proteins aid in the elucidation of the aggregation mechanism and suggest
therapeutic strategies. We have constructed phage display libraries enriched in binders
to amyloidogenic intrinsically disordered proteins, using ZAb3, a protein with high
affinity for the amyloid-beta peptide, as a scaffold. Binding proteins selected from
these libraries are termed beta-wrapins (beta-wrap proteins). The beta-wrapins AS69
and HI18 exhibit nanomolar affinity for monomeric alpha-synuclein or islet amyloid
polypeptide, respectively. AS69 and HI18 potently inhibit in vitro amyloid formation
and toxicity at substoichiometric concentration ratios, indicating that they interfere
with the nucleation and/or elongation of amyloid fibrils. The NMR structures of the
beta-wrapin:target complexes reveal beta-hairpin motifs in alpha-synuclein and islet
amyloid polypeptide which are stabilized by coupled folding and binding. In the case
of alpha-synuclein, the beta-hairpin is formed in the sequence region 35-59 which
contains the beta-strand segments b1 and b2 of amyloid fibril models and most disease-related
mutations. We show by disulfide engineering, biophysical techniques, and cell viability
assays that intramolecular tertiary interactions between the b1 and b2 segments of
alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of
amyloid-beta peptide and islet amyloid polypeptide. Our results reveal a common preference
of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate
a critical role of hairpin conformers in the control of amyloid formation.
PF-009
Interaction Profiling through Proteomic Peptide Phage Display
Cecilia Blikstad1, Moon-Hyeong Seo2, Norman Davey3, Roland Arnold2, Sachdev S Sidhu2,
Philip M Kim2, Ylva Ivarsson1
1Department of Chemistry - BMC, 2Donnelly Centre
A considerable part of the human proteome is intrinsically disordered. The disordered
regions are enriched in short motifs serving as docking sites for peptide binding
domains. Domain-motif interactions are crucial for the wiring of signaling pathways.
These interactions are typically transient and difficult to capture through most conventional
high-throughput methods. We therefore developed a novel approach for the large-scale
profiling of domain-motifs interactions called Proteomic Peptide Phage Display (ProP-PD)
(1). In ProP-PD we combine bioinformatics, oligonucleotide arrays, peptide phage display
and next-generation sequencing. This allows the interrogation of domain-motif interactions
on a proteome-wide scale and the de novo motif discovery.In our pilot experiment we
generated two distinct phage libraries, one displaying all human C-terminal sequences
and one displaying C-termini of known virus proteins. We used the ProP-PD libraries
to identify interactions of human postsynaptic density 95/discs large/zonula occludens-1
(PDZ) domains. We successfully identified novel PDZ domain interactions of potential
relevance to cellular signaling pathways and validated a subset of interactions with
a high success rate. Recently, we created a ProP-PD library that displays peptides
representing the disordered regions of the human proteome. We validate our disorderome
library against a range of peptide binding domains, which provides novel insights
into their binding preferences and suggest interactions of potential biological relevance
as will be presented here. ProP-PD can be used to uncover protein-protein interactions
of potential biological relevance in high-throughput experiments and provides information
that is complementary to other methods. ProP-PD is scalable and can be developed to
any target proteome of interest.
1. Ivarsson, Y., Arnold, R., McLaughlin, M., Nim, S., Joshi, R., Ray, D., Liu, B.,
Teyra, J., Pawson, T., Moffat, J., Li, S., Sidhu, S. S., & Sidhu, S. S. Large-scale
interaction profiling of PDZ domains through proteomic peptide-phage display using
human and viral phage peptidomes. (2014) Proc Natl Acad Sci U S A 111, 2542-2547.
PF-010
Biophysical characterization of phosducin and its complex with the 14-3-3 protein
Miroslava Kacirova1,2, Jiri Novacek3, Petr Man1,4, Alan Kadek1,4, Veronika Obsilova2,
Tomas Obsil1,2
1Faculty of Science, Charles University in Prague, 2Institute of Physiology, Czech
Academy of Sciences, 3Masaryk University, CEITEC – Central European Institute of Technology,
4Institute of Microbiology, Czech Academy of Sciences
Phosducin is a 30 kDa phosphoprotein that regulates visual signal transduction by
interacting with the Gtβγ; subunit of the retinal G-protein transducin. The function
of Pdc is regulated by phosphorylation at Ser54 and Ser73 in a process that involves
the binding of phosphorylated Pdc to the regulatory 14-3-3 protein, but the molecular
mechanism of the regulation by 14-3-3 protein is still unknown. Pdc was also suggested
to be involved in transcriptional control, the regulation of transmission at the photoreceptor-to-ON-bipolar
cell synapse, and the regulation of the sympathetic activity and blood pressure [1-3].
Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were
investigated using small angle X-ray scattering, circular dichroism, quenching of
tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange
coupled to mass spectrometry and nuclear magnetic resonance. We show that the 14-3-3
protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding
interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the
14-3-3-depedent inhibition of Pdc function. The 14-3-3 protein dimer interacts with
Pdc using surfaces both inside and outside its central channel. The N-terminal domain
of Pdc, where both phosphorylation sites and the 14-3-3 binding motifs are located,
is intrinsically disordered protein which remains likely highly flexible when bound
to 14-3-3 indicating the fuzzy-like character of this complex. In addition, it has
been speculated that the 14-3-3 protein binding decreases the rate of Pdc dephosphorylation
after a light stimulus through its interaction with phosphorylated Ser54 and Ser73,
thus lengthening the time that Pdc remains phosphorylated after a light exposure.
Pdc is dephosphorylated in vivo by protein phosphatases 1 (PP1) and 2A (PP2A). Our
dephosphorylation experiments with PP1 revealed that the 14-3-3 protein does slow
down the dephosphorylation of doubly phosphorylated Pdc in vitro.
1. R. Gaudet, A. Bohm, P. B. Sigler, Cell 87, (1996), 577-588.
2. B. Y. Lee, C. D. Thulin, B. M. Willardson, J. Biol. Chem. 279, (2004), 54008-54017.
3. L. Rezabkova, M. Kacirova, M. Sulc, P. Herman, J. Vecer, M. Stepanek, V. Obsilova,
T. Obsil, Biophysical J. 103, (2012), 1960-1969.
This work was supported by the Czech Science Foundation (Project P305/11/0708), Grant
Agency of Charles University in Prague (Project 793913); and Czech Academy of Sciences
(Research Projects RVO: 67985823 of the Institute of Physiology).
PF-011
Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1
Can Kayatekin1, Kent Matlack1, William Hesse2, Yinghua Guan3, Sohini Chakrabortee1,
Gregory Newby2, Jenny Russ4, Erich Wanker4, Jagesh Shah3, Susan Lindquist1,2,5
1Whitehead Institute For Biomedical Research, 2Massachusetts Institute of Technology,
3Harvard Medical School, 4Max Delbrück Center For Molecular Medicine, 5Howard Hughes
Medical Institute
Huntington’s disease (HD) is a devastating neurodegenerative disorder caused by an
increase in the length of a polyglutamine repeat (polyQ) in the protein huntingtin.
At least nine other proteins are also known to cause neurodegenerative disease in
a polyglutamine-length dependent manner. Despite intense study, the molecular basis
of polyQ toxicity in HD or any of the other diseases has only partially been elucidated
and potential routes to therapeutic intervention are sparse. The use of genetically
tractable model organisms to identify the cellular pathologies caused by mutant huntingtin
expression is essential to our understanding of the disease pathology in humans. In
eukaryotes, many of the protein folding homeostasis pathways are highly conserved
and yeast cells expressing a glutamine-expanded fragment of huntingtin exon 1 exhibit
a polyQ length-dependent toxicity that recapitulates many of the basic protein folding
defects associated with polyQ diseases in neurons. Taking an unbiased approach, we
screened an overexpression library of the entire yeast genome for suppressors and
enhancers of polyQ toxicity and identified seven proteins with prion-like, Q-rich
domains that are strong suppressors in yeast. Intriguingly, the Q-rich domains of
these proteins, and several other Q-rich domains, suppress toxicity when expressed
in isolation. These suppressors are also efficacious in mammalian cells and, strikingly,
one suppressor was independently shown to alleviate polyQ-expanded ataxin-3 toxicity
in a Drosophila model. In yeast, the suppressors co-aggregated with an otherwise highly
toxic 103-glutamine expanded huntingtin exon 1 protein (Htt103Q), resulting in a non-toxic
aggregate and eliminating populations of diffusible oligomeric species. Using a transcriptional
sensor for protein co-aggregation, we determined that yeast and human proteins that
normally co-aggregated with Htt103Q did not co-aggregate with these hetero-aggregates.
Thus, these Q-rich domains may suppress Htt103Q toxicity by two complementary mechanisms:
trapping potentially toxic oligomers in larger aggregates and by limiting the interactome
of the larger Htt103Q aggregates.
PF-012
Structuring disorder: the case of the intrinsically disordered Unique domain of c-Src
Mariano Maffei1
1BioNMR lab - Faculty of Organic Chemistry - University of Barcelona
About two thirds of eukaryotic proteins contain large intrinsically disordered regions.
They represent a change of paradigm from “structure-function” to “information-function”
(Uversky, 2011; Babu et al., 2011). Structured proteins are information rich, but
the current challenge is to discover how information is stored in disordered protein.
Regulation of c-Src activity, the first discovered oncoprotein, by its intrinsically
disordered N-terminal region has been recently demonstrated (Perez et al., 2013).
Functional studies have revealed that mutations in the ULBR cause strong phenotypes
when introduced in full-length c-Src and expressed in Xenopus laevis oocytes (Perez
et al., 2013) or in human SW620 colorectal cancer cells (unpublished). However, the
connection with the classical regulatory mechanisms is still missing. c-Src domain
structure consists of four “Src-homology” domains: SH4, SH3, SH2 and SH1, arranged
in this order from the N-terminus to the C-terminus, with the intrinsically disordered
“Unique” domain separating the SH4 and SH3 domains. Classically, the SH3 and SH2 domains
are involved in regulation and the SH4 domain is the membrane anchoring site. We will
present our recent results showing that the Unique domain is part of a long loop closed
by the interaction of the SH4 and SH3 domains (Maffei et al., 2015). The conformational
freedom of this disordered region is further restricted through direct contacts between
the RT-loop of the SH3 domain and, primarily, residues located within the recently
discovered Unique lipid binding region (ULBR). The interaction between the Unique
and SH3 domains is allosterically modulated by a poly-proline ligand binding to the
canonical binding site of the SH3 domain (Maffei et al., 2015). These results demonstrate
a direct connection between classical c-Src regulation involving the SH3 domain and
the new regulation mechanisms involving the intrinsically disordered regions and provide
new evidence of the functional importance and the underlying mechanism behind regulation
of signalling pathways by intrinsically disordered domains.
1. Uversky VN. Intrinsically disordered proteins from A to Z. Int J Biochem Cell Biol
43: 1090–1103 (2011).
2. Babu MM, van der Lee R, de Groot NS, Gsponer J. Intrinsically disordered proteins:
regulation and disease. Curr Opin Struct Biol 21: 432–440 (2011).
3. Pérez, Y., Maffei, M., Igea, A., Amata, I., Gairí, M., Nebreda, A.R., Bernadó,
P., and Pons, M. Lipid binding by the Unique and SH3 domains of c-Src suggests a new
regulatory mechanism. Sci. Rep. 3, 1295 (2013).
4. Maffei, M., Arbesú, M., Le-Roux, A.L., Amata, I., Roche S. And Pons, M. The SH3
domain acts as a scaffold for the N-terminal intrinsically disordered regions of c-Src.
Structure (2015), http://dx.doi.org/10.1016/j.str.2015.03.009.
PF-013
The Yeast GRASP Grh1 displays features of an Intrinsically Disordered Protein
Raquel Fonseca-Maldonado1, Felipe Mendes1, Luana Meleiro1, Assuero Garcia1, Antonio
Costa-Filho1
1Departamento de Física, Universidade de São Paulo-FFCLRP, 2Departamento de Química,
Universidade de São Paulo-FFCLRP
In mammalian cells, the Golgi reassembly and stacking proteins (GRASP55 and GRASP65)
are involved in the stacking of Golgi apparatus cisternae and in the formation of
the Golgi ribbon. Since GRASPs have been identified in many organisms, other roles
for GRASPs have already been pointed out, such as chaperoning and transport of other
proteins, involvement in cell apoptosis, cell migration, unconventional secretion,
and in mitosis. In Saccharomyces cerevisiae, it is observed that only 40% of the Golgi
cisternae are in stacks and do not form ribbon structures. This build yeast contains
a single GRASP, called Grh1, that is analogue to GRASP65. The structural differences
of the Golgi apparatus and the functional repertoire of GRASPs suggest a structural
dynamic of these proteins. Here, we used a combination of biophysical/biochemical
methods to investigate the behavior of Grh1. Bioinformatics and circular dichroism
(CD) analyses of Grh1 indicated a high percentage of either flexible regions or extended
loops. The partial unfolded Grh1 structure in solution folded into more ordered structures
under temperature increasing, dehydration onto a surface and nonaqueous solvents as
reported also by CD. Hydration of the dehydrated folded protein is a reversible process
that is accompanied by unfolding. Furthermore, Grh1 showed slow migration in SDS–PAGE,
high susceptibility to proteases and low cooperativity of the chemical-induced unfolding
process. Fluorescence of Trp residues along with CD data showed Grh1 preserves a considerable
amount of residual secondary structure, and the unfolding transition monitored by
Trp presented higher cooperativity. Another cooperative transition was also reported
by the extrinsic hydrophobic fluorescence probe ANS upon chemical denaturation. These
set of experiments indicate that Grh1 behaves as a protein containing intrinsically
disordered regions (IDRs), characterized by unstructured regions of high polypeptide
mobility experiencing many conformations. These findings suggest that an IDP-like
behavior may be the solution found by Nature to account for Grh1 functional need for
interactions with several different partners in the cell.
PF-014
Conformational changes governing dengue virus capsid protein function and its inhibition
by pep14 23
André F. Faustino1, Gabriela M. Guerra1, Roland G. Huber2, Axel Hollmann1, Peter J.
Bond2, Miguel A.R.B. Castanho1, Andrea T. Da Poian3, Fábio C.L. Almeida3, Nuno C.
Santos1, Ivo Martins1
1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 2Bioinformatics
Institute, A*STAR, 3Instituto De Bioquímica Médica, Universidade Federal Do Rio De
Janeiro
ABSTRACT Dengue virus (DENV) infection affects millions of people and is becoming
a major global disease for which there is no specific treatment available. The interaction
of DENV capsid (C) protein with host lipid droplets (LDs) is essential for viral replication.
pep14-23, a peptide designed based on a DENV C intrinsically disordered conserved
region, inhibits this crucial interaction. Combining bioinformatics and biophysics
we determined pep14-23 structure and ability to bind different phospholipids, in the
context of DENV C function. pep14-23 becomes α-helical upon binding to anionic phospholipids.
Structure prediction of DENV C N-terminal intrinsically disordered region reveals
orientations that alternatively shield or expose DENV C hydrophobic pocket, supporting
a novel autoinhibitory role for this region. These findings pave the way for similar
studies to understand disordered proteins and improved peptidomimetics drug development
strategies against flaviviruses. TOPICS Intrinsically Disordered Proteins Protein-Lipid
Interactions
PF-015
Developing mechanistic insight into modulators of tau aggregation
Eri Nakatani-Webster1, Hannah Baughman1, Shaylin Higgins1, Abhinav Nath1
1Department of Medicinal Chemistry, University of Washington
The pathological self-association of microtubule-associated protein tau is implicated
in a range of neurodegenerative disorders collectively called tauopathies, perhaps
the most prominent of which are Alzheimer’s disease (AD) and chronic traumatic encephalopathy
(CTE). Tau aggregation in vitro shares many features in common with fibril formation
by other amyloid-forming proteins: a nucleation-dependent polymerization reaction
progressing via oligomeric intermediates into β-sheet-rich fibrillar aggregates, characterized
by a distinctive sigmoidal kinetic. Over the years, many investigators have advanced
our understanding of how these time-courses might best be characterized and interpreted.
In particular, elegant analytical and numerical approaches have been developed that
supersede the empirical sigmoidal equations typically used to fit fibril formation
traces. These modern approaches have enabled more rigorous insight into the mechanism
of amyloid formation, and into how small molecules, protein chaperones, and other
binding partners can modulate the process. An understanding of a modulator’s effects
on amyloid formation mechanism is necessary in order for us to predict and engineer
its effects on amyloid pathology in a biological context. A given modulator may affect
rates of primary or secondary nucleation, elongation, or fibril fragmentation to different
extents. Each of these perturbations, individually or in combination, can alter the
kinetics of aggregation, the final state of the amyloid fibrils, and the sampled ensemble
of oligomeric intermediates. Unfortunately, fitting of mechanistic models to amyloid
formation kinetics is an example of an “ill-posed problem”, in that dramatically different
combinations of elementary parameters can nevertheless generate very similar sigmoidal
kinetic traces. This has typically necessitated global analysis of amyloid kinetic
traces collected over a broad range of protein concentrations – a substantial expenditure
of time, effort and material that must then be repeated in the presence of a modulator
in order to gain insight into its effects. We propose an alternative approach: to
fit amyloid formation traces to a large distribution of parameter sets, and determine
how various aggregation modulators affect the distribution of parameters. This so-called
“parameter distribution analysis” enables the inference of mechanistic effects from
measurements at a single protein concentration. Parameter distribution analysis based
on numerical modeling has been made tractable by advances in computer hardware and
software, and can be easily extended to include additional mechanisms or phases relevant
to a protein or modulator of interest. Here, we illustrate how parameter distribution
analysis, complemented by fluorescence correlation spectroscopy (FCS), electron microscopy
(EM) and other biochemical techniques, can shed light on fundamental aspects of tau
amyloidogenesis. We examine the disparate effects that natural products, pharmacotherapies
and protein chaperones can have on the mechanism of aggregation, and also discuss
the effects of heparin (widely used as an inducer of tau aggregation). These insights
demonstrate the value of parameter distribution analysis as applied to amyloid formation
and other ill-posed biochemical problems.
PF-016
New insights into amyloidogenesis of Tau protein induced by enantiomers of polyglutamic
acid
Bartosz Nizynski1,2,3,4, Hanna Nieznanska2, Krzysztof Nieznanski2, Wojciech Dzwolak4
1College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences,
2Department of Biochemistry, Nencki Institute of Experimental Biology, 3Institute
of High Pressure Physics, 4Department of Chemistry, Biological and Chemical Research
Centre
New insights into amyloidogenesis of Tau protein induced by enantiomers of polyglutamic
acid. Bartosz Nizynski1,2,3,4, Hanna Nieznanska2, Wojciech Dzwolak3,4, Krzysztof Nieznanski2
1. College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences,
University of Warsaw, Warsaw, Poland; 2. Department of Biochemistry, Nencki Institute
of Experimental Biology, Warsaw, Poland; 3. Institute of High Pressure Physics, Warsaw,
Poland; 4. Department of Chemistry, Biological and Chemical Research Centre, University
of Warsaw, Warsaw, Poland.Amyloidogenesis of Tau protein leads to the formation of
amyloid fibrils (ordered fibrillar protein aggregates) which are accumulated in neurons
of central nervous system during the course of neurodegenerative diseases called tauopathies.
Studying Tau (a typical intrinsically disordered protein) amyloidogenesis has been
challenging for many reasons. Positive charge on the Tau molecule must be compensated
(e.g. in the presence of polyanions) in order to initiate the process. Heparin (glycosaminoglycan)
has been the most intensively studied charge-compensating agent in this context. On
the other hand induction of Tau aggregation by polyglutamic acid is poorly characterized.
Mechanisms responsible for the propagation of Tau conformations has become an interesting
research objective. Prion–like features of Tau amyloid can be studied in vitro also
in the seed-induced regime of aggregation. Tau amyloid seeds can act as nuclei for
amyloidogenesis. Such seeds can be obtained by fragmentation of amyloid fibrils by
means of sonication. Given that amyloidogenesis can proceed through various assembly
pathways resulting in distinct amyloid ’strains’ (self–propagating structural variants
of amyloid) we have used poly–L–glutamic acid (PLGA) and poly–D–glutamic acid (PDGA)
to direct Tau onto different amyloidogenic pathways. We have hypothesized that the
chirality of the inducers could lead to fibril polymorphism. In our studies, we have
used a recombinant human 2N4R Tau isoform. We have been using transmission electron
microscopy (TEM), sedimentation and kinetic measurment. Firstly, we have characterized
unseeded PLGA–/PDGA–induced Tau aggregation to find out that corresponding kinetics
were significantly different. Secondly, we have used sonicated fibrils to characterize
the kinetics of seeded processes. Both PLGA–/PDGA–induced amyloid seeds were able
to efficiently seed Tau aggregation in the presence of PLGA, whereas in the presence
of PDGA the aggregation was much less effective. Surprisingly, we found that PDGA–induced
amyloid seeds were able to catalyze fibrillogenesis of Tau more clearly in the presence
of soluble PLGA than in the presence of PDGA – the primary inducer. We could not induce
aggregation of Tau in the absence of polyglutamic acids which indicates that positive
charge on Tau molecules must be unconditionally compensated in order to promote amyloidogenesis.
Thirdly, using TEM we have characterized different morphologies of Tau amyloid fibrils
generated in unseeded and seeded processes. Finally, to further characterize properties
of the fibrils we have performed sedimentation experiments. Fibrils induced by PLGA,
PDGA and heparin revealed different sedimentation properties. Heparin-induced fibrils
underwent sedimentation more readily than PDGA-induced fibrils, whereas PLGA-induced
fibrils remained in the supernatant. These results indicate distinct physicochemical
properties of these fibrils. We believe that our findings will contribute to the current
understanding of the molecular dynamics of Tau amyloidogenesis.
PF-017
Self-organizing structures of alpha-synulceins and its aggregates by a coarse-grained
Monte Carlo simulation
Ras Pandey1, Peter Mirau2, Barry Farmer2
1University of Southern Mississippi, 2Air Force Research Laboratory
Alpha-synuclein (ASN) consisting of 140 residues, an intrinsically disordered protein,
is linked to such neurodegenerative diseases as Parkinson’s disease (PD) and Alzheimer
disease via toxic clumping into amyloid fibrils. We investigate the structure and
dynamics of an ASN chain as a function of temperature by a coarse-grained approach
where a residue is represented by a node. In our coarse-grained approach, a residue
is represented by a node. The basic idea is borrowed from the ’united atom’ approach
in polymer chain modeling that has been used extensively where the benefits and pitfalls
of the method is explored for decades. Such coarse-grained method has also been used
protein chain modeling in recent years (e.g. AIP Advances 5, 092502 (2015)). Although
the atomic scale structural resolution is sacrificed its specificity is captured via
a set of unique knowledge-based residue-residue interactions matrix (e.g. classic
Miyazawa-Jernigan matrix, Macromolecules 18, 534 (1985)). A number of local and global
physical quantities are analyzed such as contact map, neighborhood and mobility profiles,
mean square displacement of protein, its radius of gyration and the structure factor.
Based on the mobility profile, we are able to identify three distinct segment of ASN
along its contour, i.e. sluggish N-terminal (1-60) and C-terminal (96-140, least mobile)
separated by the central region (61-95), the non-amyloid component (NAC) with higher
mobility. Contact profile shows that the probability of intra-chain residue aggregation
(clumping) is higher in the N-terminal region than the C-terminal with least aggregation
in the NAC region. We find that the radius of gyration (Rg) decays monotonically with
the temperature, consistent with the finding of Allison et al. (JACS, 131, 18314 (2009)).
From the detail analysis of the structure factor we are able to predict the variation
of the spatial mass distribution with the temperature as the residues in ASN chain
organize and disperse by evaluating its effective dimension D. We find the protein
conforms to a globular structure (D∼3) at the low temperatures and to a random coil
(D∼2) at high temperatures which is consistent with the estimates of Uversky et al.
(J. Biol. Chem. 277, 11970 (2002)). In addition, we provide the estimates of D (3 ≥ D ≥ 2)
for the intermediate structures as the protein chain makes a transition from globular
to random coil. Questions under-investigation includes what are the effects of mutations
(e.g. β- and γ-synuclein), how does the structure of an isolated ASN chain change
in presence of many interacting protein chains, and how do they organize over the
multiple length scales? Attempts will be made to address some of these issues as the
data become available.
PF-018
Tear down the wall: dismantling the biofilm scaffold of E.coli
Cesyen Cedeno1, Nani Van Gerven1, Wim Jonckheere1, Imke Van den Broek1, Han Remaut1,
Peter Tompa1
1VIB, Structural Biology Research Center
CsgA is the major subunit of the so-called curli fiber system. This is an amyloid
structure formed in the outer membrane on E.coli and acts as a scaffold for the biochemical
machinery/matrix in the extracellular milieu (biofilms). Extracellular matrices of
this nature are robust platforms helping bacteria colonization; in this context CsgA
becomes a key target in order to break the architecture within bacterial biofilms.
Chaperones are molecular machines able to stabilize misfolding prone proteins or even
retrieve proteins trapped in non-physiological states. Here we show how ERD14 acts
as a molecular chaperone inhibiting the formation of CsgA amyloid fibers in vitro.
This work illustrates an alternative approach towards biofilm treatment at a molecular
level.
PF-019
Coupled folding and binding of transcription factors
Sarah Shammas1, Alexandra Travis1, Jane Clarke1
1Department of Chemistry, University of Cambridge
Intrinsic protein disorder is ubiquitous in transcription, particularly within transcription
factors, which frequently fold into structures upon binding to partner molecules (DNA
or protein). The coupled folding and binding reactions that take place between individual
transcription factors and the key hub co-activator proteins are crucial in determining
the expression profile of the cell, and hence its phenotype. These interactions have
been well studied by structural and equilibrium methods. Here we present mechanistic
insights into the process, gained through complementary kinetics experiments, for
the binding of five separate transcription factors to a single prototypical co-activator
(CBP KIX). The transcription factors investigated belong to cellular (cMyb, MLL, CREB,
E2A) and viral (HTLV-1 bLZ) classes. These reactions are remarkably fast; after removing
the effect of long-range electrostatic rate enhancement the association rate constant
is still approximately 2 x 107 M-1s-1, which is just above the typically quoted upper
limit for diffusion-limited reactions between pairs of proteins (105 – 106 M-1s-1),
and is also the highest such value we have found reported. This, combined with the
apparent insensitivity of the association rate to residual structure within the unbound
state, indicates that binding preceeds folding (induced fit mechanism). Interactions
between KIX and its transcription factors are additionally modulated by allostery
between its two binding sites. We investigate the basis for this, finding it to be
mediated by changes in protein flexibility.
PF-020
Alternative hit finding strategies for intrinsically disordered proteins, exemplified
by forkhead-box transcription factors
Harm Jan (Arjan) Snijder1, Maria Saline1, Tomas Jacso1, Frank Janssen1, Mattias Rohman1,
Tyrrell Norris1
1Astrazeneca R&D, Discovery Sciences, SE-431 83,Pepparedsleden 1
Forkhead box O (FOXO) proteins are emerging as key transcription factors in insulin
and glucose metabolism, regulation of immune responses, and to balance cell proliferation,
apoptosis and senescence. FOXO proteins are predicted to be intrinsically disordered
proteins (IDPs); IDPs are largely unstructured and often function as hubs mediating
multiple interactions. IDPs are considered to be largely evasive from classical small
molecule interference and lead-generation approaches, as they lack defined binding
pockets. The available methods for addressing these targets have been lagging behind
and needs to be developed to assess tractability of this target class. Here we have
evaluated the tractability of fragment screening on various domains of a Forkhead
box O member. We could confirm the intrinsically disordered character of FOXO and
used NMR screening to identify fragments that interact with FOXO. One of these fragments
was subsequently confirmed as a direct FOXO binder in 2D HSQC-NMR spectroscopy and
this fragment showed an effect in a FOXO reporter gene assay. These results demonstrate
that fragment screening may be a valuable approach for intrinsically disordered proteins
although challenges remain to expand these fragments into more potent hits in the
absence of detailed structural data.
PF-021
SDS-PAGE analysis of Aß oligomers is disserving research into Alzheimeŕs disease:
a call for ESI-IM-MS
Sílvia Vilaprinyó-Pascual1, Rosa Pujol-Pina1, Roberta Mazzucato1, Annalisa Arcella2,
Marta Vilaseca3, Modesto Orozco3, Natàlia Carulla1
1Institute for Research in Biomedicine (IRB Barcelona), 2Joint IRB-BSC Research Program
in Computational Biology, 3Mass Spectrometry Core Facility, IRB Barcelona, 4Department
of Biochemistry and Molecular Biology, University of Barcelona
The characterization of amyloid-beta peptide (Abeta) oligomer samples is critical
to advance in the field of Alzheimeŕs disease (AD). Here we report a critical evaluation
of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), extensively used in the field, and electrospray ionization
ion mobility coupled to mass spectrometry (ESI-IM-MS), an emerging technique with
great potential for oligomer characterization. To evaluate their performance, we first
obtained pure cross-linked Abeta40 and Abeta42 oligomers of specific order. Analysis
of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of
Abeta42 oligomers, thus providing flawed information on their order and distribution.
In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical
modifications and on the structure of the oligomers. Our findings have important implications
as they challenge scientific paradigms in the AD field built upon the SDS-PAGE characterization
of Abeta oligomer samples.
PF-022
Coarse-grained simulation of protein association: application to rate prediction and
implication for association mechanisms
Yinghao Wu1,
1Systems and Computational Biology, Albert Einstein College of Medicine
The kinetics of protein binding is of paramount importance for understanding cellular
functions. For instance, the binding kinetics between membrane receptors and their
ligands control the speed of signal transduction after cells are exposed to stimulation.
The experimentally measured association rates of protein binding span ten orders of
magnitude, a range that was divided into two regimes. It was proposed that a fast
association regime is limited by protein diffusion, while the other side of the spectrum
is controlled by conformational changes. Consequently, all previous simulation methods
neglected conformational changes when calculating the association rate of a diffusion-limited
regime. However, the most updated theory of protein binding suggests that a protein
remains in a pre-existing equilibrium of unbound conformations. Binding shifts the
equilibrium toward its bound state. This highlights the importance of conformational
factors for regulating protein binding. Enlightened by this conformational selection
model, we hypothesize that the conformational flexibility of protein structures regulates
association more widely than previously anticipated. We develop a new coarse-grained
model to simulate the process of protein association via the kinetic Monte Carlo (KMC)
algorithm. Each residue in this model is represented by its Cα atom and a side-chain
functional site. A simple physically based potential is used to guide the relative
diffusion of two interacting proteins. Given the size of the simulation box and the
length of the simulation, the association rate constant can be derived by counting
the frequency of dimerization among a large number of simulation trajectories. We
further designed a prediction strategy that accounts for both the conformational and
energetic factors of binding. Our method is able to predict rates of protein association
that are highly correlated with experimentally measured values. Due to the coarse-grained
feature, our model was further applied to several special cases of protein association.
In one example, we studied the binding kinetics of proteins with flexible linkers.
The interaction between thrombin and its functional inhibitor, rhodniin, was used
as a testing system. We captured the conformational changes of flexible linkers from
the all-atom molecular dynamic simulations. We found that the association with full-length
flexible rhodniin was faster than its two individual domains and that their dissociation
was more difficult, supporting a “flycasting” mechanism in which partial structures
of an intrinsic disordered protein (IDP) dock to the target first, while the remaining
segments undergo conformational searches and sequentially coalesce around the target.
In another example, we studied the binding kinetics of membrane receptors from cellular
interfaces. The interaction between membrane proteins CD2 and CD58, cell adhesion
molecules known to mediate the activation of T cells and natural killer cells, was
used as a testing system. The diffusive properties of these proteins on lipid bilayer
were captured from all-atom molecular dynamic simulations. We showed that both 3D
and 2D association rates could be simulated quantitatively with our method. The calculated
values were close to the experimental measurements. We also provided detailed analysis
of how molecular diffusions and membrane fluctuations affected 2D association.
PF-023
(Un)structure-function relationships on the UreG enzyme in the nickel-dependent urease
system
Barbara Zambelli1, Francesco Musiani1, Stefano Ciurli1
1University of Bologna, Dept. of Pharmacy and Biotechnology
Urease is an essential enzyme for many pathogens and soil microorganisms. Its activity
relies on the presence of nickel in the active site (1). The incorporation of this
metal ion into the enzyme requires the formation of a supra-molecular chaperone involving
four accessory proteins, named UreD, UreF, UreG and UreE. UreE is a metallo-chaperone
involved in nickel binding and delivery into the enzyme active site. UreG is a GTPase
essential for providing energy to the process of nickel site assembly. UreF and UreD
form a complex that regulates the GTPase activity of UreG. The present work focuses
on UreG, which exists in solution as an ensemble of inter-converting conformations
(2). This observation made this protein the firstly discovered natural enzyme with
an intrinsically disordered behavior, possibly allowing it to interact with different
protein partners, such as UreE (3,4) and UreF (5) and cofactors, such as metal ions
(6), in the urease activation network. UreG folding was studied perturbing protein
conformation with temperature and denaturants, and investigating its folding response
using circular dichroism, NMR and fluorescence (7). A combination of light scattering,
calorimetry, mass spectrometry, and NMR spectroscopy shed light on the effect of metal
ion binding onto the conformational equilibrium of UreG ensemble (8). The results
suggest that metal binding and solution conditions modulate affect the protein-protein
interactions and enzymatic activity of UreG.
(1) Zambelli et al., Acc. Chem. Res. (2011), 44 (7) pp. 520-30
(2) Zambelli et al. J. Biol. Chem. (2005), 280(6):4684-4695
(3) Merloni et al. BBA - Proteins and Proteomics (2014), 1844(9):1662-74
(4) Yang et al. J. Biol Chem. 2015, in press
(5) Fong et al. Plos Biol. 2013, 11(10), e1001678
(6) Zambelli et al. Proteins (2009), 74(1):222-239
(7) Zambelli et al. Mol. Biosyst. (2012), 8: 220-228
(8) D’Urzo et al. J. Biol. Inorg. Chem. (2014), 19(8): 1341-1354
PF-024
Molecular insights into the VPg-Pro interaction from Pepper Vein Banding Virus: Implication
in protease activity
Pallavi Sabharwal1, Rashmi Panigrahi1, Srinivas Sistala2, Savithri H S1
1Indian institute of Science, 2Wipro G E Healthcare Pvt Ltd
Nuclear Inclusion protein A- protease (NIa-pro) is a protease involved in processing
of Pepper vein banding virus (PVBV) encoded polyprotein to generate various intermediates
and mature proteins at different stages of the viral life-cycle. NIa-Pro has two domains-
N-terminal Viral protein genome linked (VPg) and the C-terminal protease domain (Pro).VPg
belongs to the group of proteins that are intrinsically disordered, but attain stable
structures upon interaction with other globular proteins. Such protein- protein interactions
have a regulatory role on the function of the interacting partners. Previously, the
influence of VPg domain on the activity of Pro was studied and it was shown that there
was a substantial increase in the protease activity upon interaction with VPg (both
in cis and in trans). In the present investigation, several deletion mutants of VPg
and NIa were constructed with a view to delineate the domain of VPg involved in interaction
with Pro. It was observed that deletion of residues from N-terminus of VPg resulted
in a decrease in the activity of Pro in cis and in trans probably because of the abrogation
of interaction between the two domains. Interaction studies using SPR (Surface Plasmon
Resonance) and ELISA confirmed that the N-terminal 22 residues of VPg are important
for interaction with Pro. The N-terminal 22 residues of VPg are a part of the disordered
region of VPg and their deletion resulted in the change in the secondary structure
of the VPg and its oligomeric state. The Ser 129 and Trp 143 residues of Pro domain
were shown to be important both for the interaction of the two domains and for the
activity of protease by mutational analysis earlier. These residues were identified
to be a part of WC loop (W143-C151) which relay the conformational changes to the
active site catalytic triad (His 46, Asp 81 and Cys 151) leading to activation. However,
mutations of these residues did not completely abolish the protease activity as well
as the interaction with VPg. Therefore, in the present study H142 and H167 which are
observed to interact with Trp143 and C151 (via non-covalent interactions) were mutated
to alanine and the H142A and H167A mutants showed a drastic reduction in the activity
of Protease. Molecular dynamics simulations of the wild type pro and the mutants revealed
that Trp 143 – His 142 – His 167 – Cys 151 interaction pathway of the wild type Pro
was disrupted in the mutants and additional residues were involved in the interaction
pathway, such alterations in the network of interactions could be responsible for
the loss of activity. However, a change in the oligomeric status of these mutants
was also observed as compared to the wild-type Pro, suggesting that these residues
are important for both the structural and functional integrity of Pro and its interaction
with VPg. Thus, these results provide a molecular insight into the VPg-Pro interactions
and the modulation of their structure and function upon mutation of residues that
are part of the interaction interface.
PF-025
A novel mutant that prevents tetramerization of amyloidogenic transthyretin protein
involved in family cardiac amyloidosis (FAC)
Priscila Ferreira1, Carolina Andrade1, Antonio Neves4, Herbert Pereira2, Fernando
Palhano1, Marcia Cruz3, Debora Foguel1
1Institute of Medical Biochemistry, Federal University of Rio de Janeiro (UFRJ), 2Phisics
Institute of São Carlos, Universitiy of São Paulo (USP), 3University Hospital CFF,
Federal University of Rio de Janeiro (UFRJ), 4Department of Microbiology, Fiocruz
Pernambuco
Transthyretin (TTR) is one of many proteins that are capable of forming amyloid fibrils
in vivo. This protein is associated with two distinct amyloidosis: Familial Cardiac
Amyloidosis (FCA) that causes a restrictive cardiomyopathy and Familial Amyloid Polyneuropathy
(FAP) that affect peripheral nerves, they are hereditary and caused by mutations in
the TTR gene. The non mutated protein can also aggregate in cardiac tissue in advanced
age patients. The diagnosis was established at University Hospital since 2008 due
to a collaborative between our group and the center of Amyloidosis Antônio Rodrigues
de Mello (CEPARM). The only mutation found in Brazil was V30M in 3 patients diagnosed
in France. Our group discovered 5 new mutation not described in Brazil and a novel
mutation not described yet A19D. The diagnosed patients are registered in Transthyretin
Amyloidosis Outcomes Survey (THAOS). The novel mutation A19D causes a severe restrictive
cardiomyopathy that is certainly related to a higher profile of aggregation observed
for this mutant if compared to others amyloidogenic mutants of TTR. Structural predictions
using a bioinformatics tool called FoldX showed that the insertion of the mutation
cause a electrostatic clash that facilitates the dissociation and aggregation of protein.
This mutant was purified heterologously and biophysical studies revealed that this
protein is a dimer and not a tetramer as commonly the TTR structure. The crystallographic
structure indicates that this mutant is structurally identical to wild type. Biophysical
studies revealed that this protein is a dimer and not a tetramer as commonly the TTR
structure. The thermodynamic stability of A19d is lower than the wild type TTR. The
aggregation profile showed us that this protein can aggregate in a higher manner and
with a fast kinetic to that observed for others amyloidogenic mutants of TTR, forming
fibers in two hours of aggregation. Heterotetramers of A19D and WT are able to aggregate
in the same fiber structure. The analysis of interface interaction of this mutant
using the PDBSum showed modifications in the profile of hydrogen bonds and non bonded
contacts. In addition the oligomers of A19D are toxic for primary culture of cardiomyocytes
from murine heart. The amyloidogenic profile displayed by this new mutant can be directly
correlated with the aggressiveness observed in the disease developed by the identified
patient. Furthermore the recent consolidation of TTR diagnosis in our university hospital
led to the identification of the rare A19D variant in a Brazilian patient, suggesting
that other new, uncharacterized mutants could be identified in the coming years.
PF-027
Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured
consensus motif
Petr Tesina1,2,3, Kateřina Čermáková4, Magdalena Hořejší 3, Milan Fábry3, Frauke Christ4,
Jonas Demeulemeester4, Zeger Debyser4, Jan De Rijck4, Václav Veverka1, Pavlína Řezáčová,3,
1IOCB AS CR, 2IMG AS CR, 3Faculty of Science, Charles University in Prague, 4KU Leuven
LEDGF/p75 protein is an epigenetic reader crucial for HIV integration and MLL1 fusion-driven
leukemia development. Its interactions with HIV integrase (HIV IN) and MLL1 are considered
an attractive therapeutic target for drug development [1]. The LEDGF/p75-MLL1-Menin
complex was structurally characterized, but only partially [2]. Using NMR spectroscopy,
we identified and mapped a novel MLL1-LEDGF/p75 interface. Colony forming assays in
MLL1-AF9+ leukemic cells expressing MLL1 interaction-defective LEDGF/p75 mutants revealed
that this additional interface is essential for leukemic transformation. Interestingly,
the newly defined interface overlaps with the binding site of known LEDGF/p75 interactor,
the HIV integrase [1]. While the pathophysiological interactions of LEDGF/p75 are
intensively studied, its physiological role remains unclear. Since LEDGF/p75 contributes
to HIV integration and leukemic transformation and has become a new therapeutic target
for drug development, it is crucial to study its physiological interactions. In addition
to HIV IN and MLL1-Menin, the LEDGF/p75 integrase binding domain (IBD) also interacts
with several other proteins [3,4]. Our recent data (manuscript accepted in Nat. Commun.)
revealed structural details of LEDGF/p75 interactions with physiological binding partners.
The interaction with the LEDGF/p75 IBD is maintained by an intrinsically disordered
IBD-binding motif (IBM) common to all known cellular partners. Based on the knowledge
of this motif, we identified and validated IWS1 as a novel LEDGF/p75 interaction partner.
The detailed mapping of physiological interaction interfaces on the IBD revealed a
notable overlap with the region involved in interaction with HIV IN. Utilizing the
structural information, we explained how HIV IN outcompetes the cellular proteins.
Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new
challenge for the development of selective interaction inhibitors.
1. Cermakova, K., P. Tesina, et al., Validation and Structural Characterisation of
the LEDGF/p75-MLL Interface as a New Target for the Treatment of MLL-Dependent Leukaemia.
Cancer Res, 2014.
2. Huang, J., B. Gurung, et al., The same pocket in menin binds both MLL and JUND
but has opposite effects on transcription. Nature, 2012. 482(7386): p. 542-6.
3. Bartholomeeusen, K., F. Christ, et al., Lens epithelium-derived growth factor/p75
interacts with the transposase-derived DDE domain of PogZ. J Biol Chem, 2009. 284(17):
p. 11467-77.
4. Bartholomeeusen, K., J. De Rijck, et al., Differential interaction of HIV-1 integrase
and JPO2 with the C terminus of LEDGF/p75. J Mol Biol, 2007. 372(2): p. 407-21.
PF-028
Exploring Anti- amyloidogenic Attributes of Lantibiotic Nisin
Deovrat Begde1
1Department of Biochemistry & Biotechnology, Dr. Ambedkar College, Deekshabhoom
Amyloid fibrils result from misassembly of peptides either formed during conventional
or aberrant intracellular protein processing events. β-amyloid peptides which nucleate
Amyloid fibrils formation are well known to be responsible for neurodegenerative disorders
like Alzheimer’s. Surprisingly, mammalian system has an inbuilt program for critical
regulation of synthesis of amyloid fibrils during formation of melanosomes. Similarly,
pathogenic bacteria like E. coli and Salmonella are known to produce extracellular
curli fimbrae, which has all the hallmarks of a typical amyloid and presumably necessary
for host colonization and virulence. Here we investigate a unique mechanism of virulence
attenuation employed by Lantibiotic, Nisin, which is a autoinducer pheromone peptide
produced by a probiotic bacterium Lactococcus lactis to selectively exterminate other
competitive Gram-positive bacteria. The preliminary study results gave promising evidences
about the exceptional ability of nisin to interfere with cell-cell communication of
selected Gram negative opportunistic pathogens, whereby it appeared to create a chaos
perhaps attributing towards swarming inhibition and biofilm disruption. Nisin caused
a disturbance in expression of virulence factors in Proteus mirabilis, Pseudomonas
aeruginosa and Escherichia coli, possibly through abolishment in expression of Gram
negative signaling molecule, homoserine lactone (HSL). Nisin demonstrated an unusual
competitive inhibition of E. coli curli assembly besides downregulation of HSL production
which regulates curli biogenesis in E. coli population. When curli positive E. coli
clinical isolates were treated with nisin their ability to form curli fimbrae was
significantly reduced. An in silico docking analysis revealed remarkable similarity
in the interaction motifs involved in curli nucleator protein, Csg B interaction with
nisin compared to those required for curli structural protein, Csg A and Csg B interaction.
Based on our results we hypothesize that nisin might block the access of Csg A to
the amyloidogenic domain of Csg B by interacting with the residues necessary for Csg
A interaction with Csg B. Although our prediction needs validation, the present investigation
provides sufficient evidences to demonstrate high anti-virulent potential of nisin
against Gram negative bacteria which are known to be eluded from its anti-microbial
spectrum.
Keywords:
Lantibiotics, Nisin, Curli, Amyloid, Anti-virulence, Homoserine lactone
PF-029
Amyloidogenic lysozyme accumulates in the endoplasmic reticulum tangling with GRP78/BiP
and evokes ER stress
Yasushi Sugimoto1, Yoshiki Kamada1, Yusuke Nawata1, Takahiro Kusakabe2
1Kagoshima University, The United Graduate School of Agri. Sci., 2Kyushu University,
Graduate School of Agri. Sci.
Naturally occurring single mutants, I56T, F57I, W64R and D67H of lysozyme in human,
have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate
in several organs, including liver, spleen and kidney, resulting in familial systemic
amyloidosis. These human pathogenic lysozyme variants are considered to raise subtle
conformational changes compared to the wild type. Here we examined the effects of
the aberrant mutant lysozymes I56T, F57I,W64R and D67H, each of which possesses a
point mutation in its molecule, on a cultured human cell line, HEK293, in which the
genes were individually integrated and overexpressed. Western blot analyses showed
lesser amounts of these variant proteins in the medium compared to the wild type,
but they were abundant in the cell pellets, indicating that the modified lysozyme
proteins were scarcely secreted into the medium but were retained in the cells. Immunocytochemistry
revealed that these proteins resided in restricted regions which were stained by an
endoplasmic reticulum (ER) marker. Moreover, the overexpression of the mutant lysozymes
were accompanied by marked increases in XBP1s and GRP78/BiP, which are downstream
agents of the IRE1_ signaling pathway responding to the unfolded protein response
(UPR) upon ER stress.RNAi for the mutant lysozymes’ expression greatly suppressed
the increases of these agents. Next, we addressed the interaction between amyloidogenic
lysozyme and GRP78/BiP as the former proteins were obtained by immunoprecipitation
with the latter protein as well as colocalization of both proteins in the ER. Lysozyme
composes of α-domain rich in helices and β-domain rich in sheet. Two helices of α1
and α2 in the N-terminal region arrange in parallel and face to face where hydrophobic
amino acids at the 3F, L8, L12, L15, L25 and L31 allocate with equal interval there.
In the back of dock, there is a core region of amyloid fibril formation, of which
the side chain of I56 is exposed on the protruding. Probably, these hydrophobic amino
acids might be crucial for lysozyme folding. Although mutated lysozymes undergo folding
by GRP78/BiP in such environment, the dissociation of the GRP from lysozyme by failure
of folding is likely inhibited and both proteins remain bound to, resulting in staying
to the ER. A part of aberrant lysozymes seem to remain bound to GRP78/BiP during folding
and insolubilize with aggregation, thus accumulate in the ER accompanied with ER stress.
Lysozyme amyloidosis might be caused by long-term accumulation in the endoplasmic
reticulum of the abnormal protein.
PF-030
Structural characterization of toxic oligomers that are kinetically trapped during
alpha-synuclein fibril formation
Serene W. Chen1, Srdja Drakulic2, Emma Deas3, Myriam Ouberai4, Francesco A. Aprile1,
German Rivas5, Andrey Y. Abramov3, Jose Maria Valpuesta2, Christopher M. Dobson1,
Nunilo Cremades1
1Department of Chemistry, University of Cambridge, CB2 1EW, 2Department of Macromolecular
Structure, Centro Nacional de Biotecnologia, 28049, 3Department of Molecular Neuroscience,
University College London, WC1N 3GB, 4Nanoscience Centre, Department of Engineering,
University of Cambridge, CB3 0FF, 5Department of Cellular and Molecular Biology, CIB-CSIC
The accumulation of abnormally aggregated proteins within the body is a common feature
of several medical disorders, such as Alzheimer’s disease, Parkinson’s disease and
diabetes mellitus type 2. While the specific protein found to be the major component
of such deposits varies from one disease to another, the formation of the pathological
aggregates seems to occur via a common process of misfolding and self-assembly of
a normally soluble polypeptide chain into a series of oligomeric intermediates and,
ultimately, into insoluble amyloid fibrils that accumulate within specific organs
and tissues. Increasing evidence indicates that certain oligomeric protein species
generated during the self-assembly of specific proteins into ordered fibrillar aggregates
can be highly cytotoxic and are likely to be key players in the initiation and spreading
of neurodegenerative diseases. However, little detailed structural information is
currently available for these oligomeric species due to their often transient nature
and, more importantly, because of their variability in terms of size and structure.
We report here the isolation and detailed characterization of an ensemble of stable
toxic oligomers of alpha-synuclein, the protein whose deposition is the hallmark of
Parkinson’s disease. By defining and minimizing the degree of heterogeneity of these
isolated alpha-synuclein oligomers which have accumulated during the process of amyloid
formation, we have identified distinct subgroups of oligomers and determined their
structural properties and three-dimensional molecular architectures. This characterization
has been achieved by the application of a set of complementary biophysical techniques,
including a variety of spectroscopic techniques along with analytical ultracentrifugation,
atomic force microscopy, and electron microscopy. Although these oligomers exist in
a range of sizes, with different extents and nature of beta-sheet content and exposed
hydrophobicity, all the oligomeric subgroups possess hollow cylindrical architectures
with marked similarities to amyloid fibrils. This suggests that these types of oligomers
are kinetically trapped during protein self-assembly and that the accumulation of
at least some forms of amyloid oligomers is likely to be a consequence of very slow
rates of rearrangement of their beta-sheet structures. Our findings reveal the inherent
multiplicity of pathways of protein misfolding and the key role the beta-sheet geometry
acquired in the early stages of the self-assembly process plays in dictating the rates
of structural conversions, and thus the kinetic stabilities and pathological nature
of different amyloid oligomers. The results of this study provide the basis for a
more complete understanding of the nature of the self-assembly of polypeptides into
beta-sheet rich amyloid aggregates, and potentially contributes to efforts to identify
specific targets for drug discovery.
Chen SW, et al. (2015) Proc Natl Acad Sci USA 112(16):E1994-E2003
PF-031
Metal ions modulate the conformation of Starmaker-like protein from Oryzias latipes
Mirosława Różycka 1, Magdalena Wojtas1, Natalie Mutter2, Benjamin Schuler2, Jacek
Gapiński3,4, Andrzej Ożyhar1
1 Wrocław University of Technology, Department of Biochemistry, 2University of Zurich,
Department of Biochemistry, 3Molecular Biophysics Department, Faculty of Physics,
Adam Mickiewicz University, 4NanoBioMedical Center, Adam Mickiewicz University
Fish otoliths and mammalian otoconia, biominerals composed of calcium carbonate and
organic matrix, are involved in the functioning of the inner ear, the sensory organ
that plays an important role in hearing and balance [1]. However, their developmental
origins, growth, and the role of the matrix, especially the protein component, are
still poorly understood. It has been shown that proteins involved in the formation
of biominerals are usually very acidic. They often belong to the group of intrinsically
disordered proteins (IDPs), a class of proteins devoid of a rigid tertiary structure
[2, 3]. The shape and polymorph selection of calcium carbonate otolith in Danio rerio
is controlled by the Starmaker (Stm) protein [4]. Recently, a gene was identified
encoding the Starmaker-like (Stm-l) protein from Oryzias latipes, a putative homologue
of Stm. It has been suggested that Stm-l has a similar function as Stm, although there
is no sequence similarity between Stm and Stm-l [5]. Several methods, such as size
exclusion chromatography, CD spectroscopy and analytical ultracentrifugation demonstrated
that Stm-l is an coil-like IDP, with the tendency to form locally ordered structures
[6]. Because Stm-l was suggested to play a crucial role in calcium carbonate mineralization,
it is possible that calcium ions may influence its conformation, as was previously
shown for Stm [7]. However, other ions may also be involved in this process. The aim
of this study was to investigate the effect of mono and divalent metal ions on the
conformation of Stm-l. We used single molecule Förster resonance energy transfer (smFRET)
and fluorescence correlation spectroscopy (FCS), which have shown that calcium ions
compacts the proteins most efficiently, followed by magnesium and the monovalent ions.
The difference in the effect of monovalent and divalent ions on the protein dimensions
is likely to result from the different properties of the ions, like charge density
and radius. CD experiments have shown that a high excess of calcium ions caused the
formation of ordered secondary structure in Stm-l, which may be crucial for the formation
of calcium carbonate crystals, when the ratio of building ions to protein is high.
This work was supported by the statutory activity subsidy from the Polish Ministry
of Science and Higher Education for the Faculty of Chemistry of the Wroclaw University
of Technology. The cost of participation was covered by Wroclaw Centre of Biotechnology,
programme The Leading National Research Centre (KNOW) for years 2014-2018.
[1] L. Addadi, et al. Z.Kardiol., 2001, 90: 92-8.
[2] M. Wojtas, et al. Advanced Topics in Biomineralization. Jong Seto (Ed.) 2012,:
3-32.
[3] V.N. Uversky. Eur.J.Biochem., 2002, 269 (1): 2-12.
[4] C. Sollner, et al. Science, 2003, 302 (5643): 282-6.
[5] B. Bajoghli, et al. Dev.Dyn., 2009, 238 (11): 2860-6.
[6] M. Rozycka, et al. PLoS One, 2014, 9 (12): e114308.
[7] T.M. Kaplon, et al. Biochim.Biophys.Acta, 2009, 1794 (11): 1616-24.
PF-032
Intrinsically disordered recombinant 57K fragment of human DMP1 influences the in
vitro crystallization of CaCO3
Aleksandra Porebska1, Andrzej Ozyhar1, Piotr Dobryszycki1
1Wroclaw University of Technology, Department of Biochemistry
Biomineralization refers to a wide range of processes by which living organisms form
mineral crystals. Usually those crystals are formed and deposited for different purposes
within the organic matrix and vesicles. Otoliths in bony fishes and otoconia in mammals
are composite crystals consisting of calcium carbonate. These biominerals are part
of the gravity and linear acceleration detection system of the inner ear. CaCO3 from
otoconia of different species have various morphology and crystalline structure and
different protein composition what underlines the importance of proteins for the proper
otoconia formation. Dentin matrix protein 1 (DMP1) is a noncollagenous protein of
extracellular matrix predominately expressed in bone and dentin. DMP1 plays an important
role in proper phosphate homeostasis and mineralization. It has been demonstrated
that DMP1 is proteolytically processed into fragments, including 37K N-terminal region
and 57K C-terminal region. As many proteins characterized to be engaged in biomineralization,
DMP1 and its fragments belong to the group of intrinsically disordered proteins (IDPs).
It has been suggested that DMP1 and its fragments can take a part in otoconia mineralization,
as the protein is present in mouse otoconia, but the role of DMP1 and its fragments
in the mineralization of calcium carbonate has not been examined until now. To determine
the influence of the DMP1 fragments for otoconia development, 57K DMP1 protein was
expressed in bacterial expression system, purified and used in in vitro biomineralization
test of calcium carbonate. In particular, immobilized metal anion affinity chromatography
(IMAC) was applied as a first step of purification procedure. Because of high content
of acidic amino acids, ion exchange chromatography with a Mono Q column was used as
a next step. Pure protein was tested in in vitro mineralization test which demonstrated
that 57K DMP1 influences size and shape of calcium carbonate crystals. Raman spectroscopy
revealed the calcite polymorph of crystals obtained in the presence of 57K DMP1. Circular
dichroism spectra showed the intrinsic disorder of 57K protein. Stokes radius estimated
based on size exclusion chromatography of protein suggested an extended conformation
of protein. Thus, 57K DMP1 may play a role in biomineralization of otoconia.
Acknowledgement:
This work was supported by the statutory activity subside from the Polish Ministry
of Higher Education for the Faculty of Chemistry of Wroclaw University of Technology.
The costs of participation were covered by Wroclaw Centre of Biotechnology, programme
The Leading National Research Centre (KNOW) for years 2014-2018.
PF-033
Structural analysis of the C-terminal domain of Drosophila melanogaster Methoprene
tolerant protein (Met)
Marta Kolonko1, Katarzyna Ożga1, Rafał Hołubowicz1, Andrzej Ożyhar1, Beata Greb-Markiewicz1
1Wroclaw University of Technology
The development of insects is regulated by the combined action of ecdysteroids and
juvenile hormones (JH). Pulses of 20-hydroxyecdysone (20E) initiate each step of metamorphosis,
while JH modulates its action and prevents precocious differentiation. The biological
and molecular mechanism of 20E action is well described. In contrary, the way of the
JH activity is still poorly understood. In 1986 Wilson and Fabian [1] reported that
Drosophila melanogaster mutants lacking Met are resistant to toxic doses of JH and
its analogue methoprene. It has been proved, that Met binds JH at physiological conditions.
Therefore Met is believed to be a putative JH receptor. Met may also be involved in
a cross-talk between two hormonal signalling pathways, involving 20E and JH. The detailed
structure of Met is still unknown. Therefore our main aim is to characterize structural
properties of Met. In silico analysis performed on a full-length Met suggested, that
N-terminal part of Met contains three conserved domains characteristic for bHLH-PAS
transcription factors, whereas C-terminal part is most probably unstructured. For
further structural analysis, we divided Met into two fragments: the first one containing
N-terminal part with bHLH and PAS domains and the second one encompassing C-terminal
part of Met. In this presentation, the results of experiments executed for C-terminal
part of Met (MetC) are presented. The expression of MetC in a E. coli cells, at a
low temperature, enabled to obtain high amount of a soluble protein. After four steps
of purification, the samples of protein with purity suitable for further analysis
were collected. CD spectroscopy analysis indicated, that MetC is highly disordered.
After incubation with TFE (tetrafluoroethylene) it acquires secondary structure elements
consisting dominantly of α-helices. Analysis of the hydrodynamic properties of MetC
performed by analytical size-exclusion chromatography and analytical ultracentrifugation
proved that MetC is a highly elongated protein with a disordered structure. In conclusion,
the results strongly suggest that C-terminus of Met shows the characteristics typical
for intrinsically disordered proteins. The disordered tails of transcription factors
are often involved in interactions with other partners by serving as a platform for
multiple interactions. It can be hypothesised that the lack of defined structure of
MetC enables this protein to play an important role in signalling pathways. This work
is supported by a statutory activity subsidy from the Polish Ministry of Science and
Higher Education for the Faculty of Chemistry of Wrocław University of Technology.
Attending in the Conference is supported by Wroclaw Centre of Biotechnology, programme
The Leading National Research Centre (KNOW) for years 2014-2018.
[1] Wilson T., Fabian J.: ‘A Drosophila melanogaster mutant resistant to a chemical
analog of juvenile hormone’; Dev Biol. 1986 Nov;118(1):190-201
PF-034
Designed cross-amyloid inhibitors of amyloid self-assembly
Eleni Malideli1, Erika Andreetto1, Li-Mei Yan1, Michael Kracklauer1, Karine Farbiarz1,
Marianna Tatarek-Nossol2, Aphrodite Kapurniotu
1Division of Peptide Biochemistry, Technische Universität München, 2Institute of Biochemistry
and Molecular Cell Biology, RWTH Aachen University
Designing inhibitors of amyloid self-assembly of intrinsically disordered polypeptides
and proteins is a difficult task. Cross-interactions between amyloidogenic polypeptides
have lately emerged as important modulators of protein self-assembly and similar surfaces
are often involved in both self- and hetero-association. We have earlier identified
a high affinity interaction between non-fibrillar and non-toxic species of Abeta and
IAPP, two intrinisically disordered, key amyloidogenic polypeptides in Alzheimer’s
disease (AD) and type 2 diabetes (T2D) (Yan et al., Angew. Chem. Int. Ed. (2007);
Andreetto, Yan et al., Angew. Chem. Int. Ed. (2010)). The Abeta-IAPP interaction results
in formation of non-fibrillar and non-cytotoxic Abeta-IAPP hetero-oligomers thus suppressing
cytotoxic self-association of both polypeptides. More recently, we have identified
short IAPP segments as “hot segments” of both IAPP self- and its hetero-association
surface with Abeta (Andreetto, Yan, et al., Angew. Chem. Int. Ed. (2010)). Capitalizing
on self- and cross-amyloid interactions, we designed highly effective, peptide-based
inhibitors of amyloid self-assembly of Abeta and IAPP. Due to their favourable properties
the designed peptides are promising leads for targeting protein aggregation in AD,
T2D or both diseases while the inhibitor design strategy should be applicable to other
amyloidogenic polypeptides and proteins as well.
PF-035
Preparation of homogenous recombinant FKBP39 protein from Tribolium castaneum
Aneta Tarczewska1, Małgorzata Kozłowska1, Andrzej Ożyhar1
1Department of Biochemistry, Faculty of Chemistry, Wrocław, University of Technolo
Juvenile hormone (JH) and ecdysteroids regulate a wide variety of developmental and
physiological processes in insects. In contrast to ecdysteroids, the exact role and
mechanism of action of JH has not been understood. Also the cross - talk between JH
and ecdysteroids remains a puzzle. Recently, common 29bp regulatory element (JHRE)
in promoter regions of some JH-responsive genes in Drosophila melanogaster, was identified.
Two proteins, immunophilin FKBP39 and calponin like Chd64 were found to bind JHRE
and interact with each other as well as other nuclear proteins including ecdysteroid
receptor, ultraspiracle nuclear receptor and methoprene tolerant protein. This suggest
that FKBP39 and Chd64 play important role in cross – talk between JH and ecdysteroid.
Researches on FKBP39 from D. melanogaster are already carried out in our laboratory
and we decided to extend our studies to distantly related species - the red flour
beetle Tribolium castaneum. Tribolium has become an important model organism for comparative
to Drosophila studies of insect development and growth. It shows classical developmental
response to JH. To facilitate the exploration of the structure function relationship
of FKBP39 we developed and optimised a protocol for its efficient expression and purification.
To express FKBP39 recombinant plasmid vectors pQE80L-XH, pQE80L-SX, pQE80L-SXH in
fusion with His-tag (XH), Strep-tag (SH) and both of those tags (SXH) were prepared.
Solubility analysis revealed that all expressed recombinant proteins remain in the
soluble fraction of bacterial proteins. Proteins fused with His-tag and Strep-tag
were able to bint TALON® and StrepTactin resin. Finally we elaborated a two-step purification
procedure for the homogenous FKBP39 using affinity chromatography (TALON® resin) and
gel filtration. The molecular mass value (40221.86 ± 1 Da) was determined using electrospray
ionisation mass spectroscopy. The value is compatible with theoretical value (40223.7
Da). TcFKBP39 can further be used for structure – function relation studies.
ACKNOWLEDGMENTS:
The work was supported by the National Science Centre grant (2012/05/B/NZ1/00659).
Conference fee was covered by Wroclaw Centre of Biotechnology, programme The Leading
National Research Centre (KNOW).
PG-001
Live-cell Measurements of the Conformational Rearrangements in Bax at the Initiation
of Apoptosis
Robert Gahl1, Yi He1, Shiqin Yu1, Nico Tjandra1
1Biochemistry and Biophysics Center, NHLBI, NIH
Apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular
organisms to ensure proper tissue homeostasis, embryonic development and immune system
activity. The Bcl-2 family of proteins regulates the activation of apoptosis through
the mitochondria pathway. Dynamic interactions between pro- and anti-apoptotic members
of this family keep each other in check until the proper time to commit to apoptosis.
The point of no return for this commitment is the permeabilization of the outer-mitochondrial
membrane (OMM). Translocation of the pro apoptotic member, Bax, from the cytosol to
the mitochondria is the molecular signature of this event. Molecular interactions
and conformational changes associated with this event have been difficult to obtain
due to challenges associated with taking subtle measurements in the complex environment
of live cells. To circumvent these challenges, we developed a novel method to reliably
detect Förster Resonance Energy Transfer (FRET) between pairs of fluorophores to identify
intra-molecular conformational changes and inter-molecular contacts in Bax as this
translocation occurs in live cells. In the cytosol, our FRET measurements indicated
that the C-terminal helix is exposed instead of tucked away in the core of the protein.
This coincided with measurements using fluorescence correlation spectroscopy (FCS)
that showed that cytosolic Bax diffuses much slower than expected, suggesting possible
complex formation or transient membrane interaction. We propose that this exposed
helix allows for this contact to occur. Cross-linking the C-terminal helix (α9) to
helix α4 reduced the instances of these interactions while at the same time yielded
FRET measurements that are consistent with the α9 helix tucked into the core of the
protein. After translocation, our FRET measurements showed that Bax molecules form
homo-oligomers in the mitochondria through two distinct interfaces involving the BH3
domain (helix α2) and the C-terminal helix. These findings provide insight into the
molecular architecture that may involve possible contacts with other Bcl-2 proteins
to permeabilize the OMM, which would also be necessary for the regulation of apoptosis.
PG-002
Bacterial cell division in super resolution
Jie Xiao1, Carla Coltharp1, Jackson Buss1, Xinxing Yang1
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine
Current advances in fluorescence-based superresolution imaging have illustrated great
details of cellular structures that are invisible to conventional fluorescence light
microscopy. The significantly improved spatial resolution is especially advantageous
for bacterial cells because of their small sizes. In the past few years the spatial
organization and dynamics of a variety of bacterial cellular structures and protein
macromachineries have been revealed with unprecedented details. As the field matures,
it is now time to focus on the functional aspect of the observed spatial organizations
and dynamics. Are they essential in carrying out a specific cellular function? Do
they play a regulatory role in controlling the on and off of a certain cellular process?
In this work I will present a few examples from our laboratory that examine the spatial
and functional organization of macromolecules involved in bacterial cell division.
PG-003
Mapping transcription factors dynamics and interactions by advanced fluorescence microscopy
techniques
Martin Stortz1, Luciana Bruno2, Paolo Annibale3, Enrico Gratton3, Adali Pecci2, Valeria
Levi4,5
1IFIByNE-Conicet, 2IFIBA-Conicet & Dept. of Physics, University of Buenos Aires, 3LFD-University
of California, 4Dept. of Biological Chemistry, University of Buenos Aires, 5IQUIBICEN-Conicet
Transcription factors (TF) exert their function by interacting with other proteins
and binding to DNA. The nucleus is a compartmentalized space, and the spatial organization
of TFs and their partners represents other step of gene expression regulation. We
used the glucocorticoid receptor (GR) as a model of TF’s mechanism of action. GR is
a ligand-activated TF with a relevant role in physiology and a great variety of effects.
It can be recruited to specific response elements on DNA or interact with other TFs.
Also, the activity of GR is modulated by different co-regulators, e.g. TIF2/GRIP1.
GR and TIF2 do not distribute homogeneously within the nucleus but accumulate in distinctive
clusters. The functional role of this particular intranuclear organization remains
unknown. We used advanced fluorescence microscopy techniques to study the dynamics
of GR and TIF2 in the nucleus of living cells with high spatial and time resolution.
GR and TIF2 fused to fluorescent tags were transiently expressed in newborn hamster
kidney (BHK) cells and visualized by a confocal microscope. Fluorescence correlation
spectroscopy (FCS) experiments were carried on to measure the intranuclear mobility
of both proteins. The method is based on the analysis of fluorescence intensity fluctuations
due to the movement of fluorescent molecules in and out the confocal volume. The data
could be fitted with a model that considers a free diffusion of TIF2 and GR in the
nucleus and their binding to fixed targets. We also studied the dynamics of different
GR mutants in the presence of different ligands and our results suggest that the binding
depends on DNA. Both GR and TIF2 autocorrelation curves reveal an increase in the
bound population upon GR activation by its agonist dexamethasone (Dex). A cross-correlation
analysis showed that, as expected, Dex-stimulus increases the population of GR-TIF2
complexes. Without hormone, GR shows a homogeneous distribution and TIF2 forms large
clusters in the nucleus. Upon Dex-binding, GR accumulates in the nucleus, is rapidly
recruited to TIF2 foci and there is an important re-distribution of both proteins,
that co-localize in the same pattern of small intranuclear clusters. The dynamics
of GR and TIF2 molecules at these clusters were studied by performing orbital-scanning
measurements, tracking the clusters position in silico and analyzing the intensity
fluctuations of the clusters along time. A positive cross-correlation between both
channels indicates that Dex-bound GR and TIF2 interact at these foci and dissociate
from them forming TIF2-GR hetero-complexes. In conclusion, advanced fluorescence microscopy
methods allowed obtaining a dynamical map of GR distribution and function in the nucleus
of mammalian living cells.
PG-004
Assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial
prionoid RepA-WH1
Cristina Fernández1, Rafael Núñez-Ramirez1, Mercedes Jimenez1, Germán Rivas1, Rafael
Giraldo1
1Centro de Investigaciones Biológicas, CSIC
Amyloid fibril formation is associated with human neurodegenerative diseases. Prefibrillar
oligomers formed during the fibril assembly process, rather than mature fibrils are
known to be central to disease and may be responsible for cell damage. A commonly
proposed mechanism for the toxicity of small oligomers is their interaction with the
lipid bilayer of cell membranes, leading to loss of membrane integrity [1]. Recent
studies from our laboratory have shown that RepA-WH1, a winged-helix domain from a
bacterial plasmid replication protein, can assemble into amyloid fibrils in vitro.
When expressed in Escherichia coli RepA-WH1 functions as a cytotoxic protein that
shares features with the mammalian amyloid proteinopathies. These features have proved
RepA-WH1 to be a suitable synthetic model system to study protein amyloidosis [2,3,4].
In this work, using the RepA-WH1 bacterial model system, we have studied the interaction
between the protein and model membranes (large and giant unilamellar lipid vesicles,
LUVs, and GUVs respectively). RepA-WH1 shows association and aggregation to membranes
composed of anionic phospholipids. Protein association in GUVs did not result in lysis
of the vesicles, suggesting the assembly of discrete protein pores as the mechanism
for RepA-WH1 membrane damage. To investigate the formation of pores we analyzed by
electron microscopy the aggregation of RepA-WH1 in the presence of a pre-formed E.
coli lipid monolayer. The EM images show the presence of pore-like particles on the
monolayer. Amyloid pores formation explains the permeabilization effect of RepA-WH1
in vesicle models and is in agreement with observations for human amyloidogenic proteins.
The approaches presented here provide a deeper insight into amyloid cytotoxicity towards
membranes and will make possible the assay of inhibitors and effectors of amyloidosis
under controlled conditions.
References:
[1] Butterfield and Lashuel. Angew Chem Int Ed. 2010; 49:5628-5654.
[2] Giraldo, et al. Prion 2011; 5:60-64.
[3] Gasset-Rosa, et al. Mol Microbiol. 2014; 91:1070-1087.
[4] Fernández-Tresguerres, et al. Mol Microbiol. 2010; 77:1456-1469.
Sponsored by Jody McGinness (jmcginness@proteinsociety.org)
PH-001
Investigation of allosteric communication pathways in human ß2-adrenergic receptor
Basak Akdas1, Ozge Kurkcuoglu2, Pemra Doruker1, Demet Akten3
1Department of Chemical Eng. and Polymer Research Center, Bogazici University, 2Department
of Chemical Engineering, Istanbul Technical University, 3Department of Bioinformatics
and Genetics, Kadir Has University
β2-adrenergic receptor (β2AR) is a member of G protein-coupled receptors, which represent
the single largest family of cell surface receptors involved in signal transduction.
β2AR recognizes a variety of ligands and communicates with cytoplasmic G-proteins
by transmitting signals through the cellular membrane. Thus, investigation of communication
pathways for β2AR may give important insights for understanding its allosteric mechanisms
and identifying new target sites for more specific and efficient drug molecules to
be used in the treatment of pulmonary and cardiovascular disease. In this study, various
conformations from 2 µs molecular dynamics (MD) simulations and available crystal
structures of human β2AR were investigated to reveal alternative signaling pathways
between its extra and intracellular regions. Specifically, shortest communication
paths connecting key residues (more than 35 Å apart) at the orthosteric ligand binding
site (D113, S203, T286, F289, N312) to either L266 or S329 located near the G-protein
binding site were investigated. The conformers from previous MD simulations[1] include
the intracellular loop 3 (ICL3), which especially affects the transmembrane collective
dynamics but is lacking in x-ray structures. The protein was described as a graph
composed of nodes linked by edges. Nodes were placed at the alpha-carbon atoms and
the edges were calculated based on the number of atom-atom interactions within a cut-off
distance 4.5 Å for each residue pair. Twenty shortest pathways were revealed using
k-shortest path algorithm[2] on the coarse-grained network. Our results indicated
that distinct signaling paths progressed most frequently on TM6 but alternative paths
were also present, which passed partially through TM5, TM7, TM3 or TM2 depending on
the conformation. Among the critical residues that transmitted the signal between
distant sites, F282 and N318 were detected, whose functional roles were reported in
previous experimental studies. Pathway shifting was observed depending on the open-to-closed
transition of ICL3 during MD simulations.
[1] Ozcan, O., Uyar, A., Doruker, P., Akten, E.D. Effect of intracellular loop 3 on
intrinsic dynamics of human β2-adrenergic receptor. BMC Structural Biology, 2013;
13:29.
[2] Yen, J. Y. Finding the k shortest loopless paths in a network. Management Science,
1971; 17, 712.
PH-002
Neonatal diabetes and congenital hyperinsulinism mutations change molecular interactions
in SUR1 NBD1
Claudia Alvarez1,2, Marijana Stagljar2, Voula Kanleis1,2,3
1Department of Chemistry, University of Toronto, 2Department of Chemical and Physical
sciences, University of Toronto, 3Department of Chemical and Physical sciences, University
of Toronto
The sulfonylurea receptor 1 (SUR1) is an ATP binding cassette (ABC) protein that forms
the regulatory subunit in KATP channels found in the pancreas and the brain. MgATP
binding and hydrolysis at the two cytosolic nucleotide binding domains (NBD1 and NBD2)
in SUR1 control gating of the KATP channel pore. 1,2 Proper regulation of KATP channel
gating by SUR1 is critical. 2 Over 100 mutations that lead to diabetes, hyperinsulinism
and developmental delay have been identified in different domains of SUR1, including
the NBDs. 3 Therefore, molecular-level understanding of the structure and function
of the NBDs is essential for designing improved treatments for SUR-related diseases.
Here we present biophysical and biochemical studies aimed at understanding the effect
of disease-causing mutations on the conformation and nucleotide binding of SUR1 NBD1.
Specifically, we are investigating SUR1 NBD1 mutations that cause neonatal diabetes
(R826W and H863T) or congenital hyperinsulinism (C717 Δ, G716V, R824G, R837 Δ and
K890T). 3 Our nuclear magnetic resonance (NMR) data shows that the hyperinsulinism
mutation K890T causes chemical shift changes throughout the spectrum of NBD1, implying
overall changes in protein conformation that may affect MgATP binding and inter-domain
interactions with other domains in the SUR1 protein. Size-exclusion data show that
the other hyperinsulinism mutations (C717 Δ, G716V, R824G, R837 Δ) produce mostly
aggregated protein, likely as a result of misfolding of NBD1. Misfolding of NBD1 may
be the underlying cause of reduced KATP trafficking seen with these mutations and
hence decreased KATP channel gating observed in hyperinsulinism. In contrast to the
K890T mutations, the congenital diabetes-causing mutations (R826W and H863T) cause
few NBD1 NMR spectral changes. However, the congenital diabetes mutation R826W decreases
the affinity of NBD1 for MgATP, which is unexpected for congenital diabetes mutations.
Our fluorescence, circular dichroism and microscale thermophoresis data corroborate
the results that we have obtained by NMR spectroscopy. Our data provide molecular-level
details on the effects of disease causing mutations in human SUR1.
1. Campbell, J.D. et al.(2003) Embo Reports 4(11): 1038-104
2. Nichols, C. G. (2006). Nature 440(7083): 470-476 3. Lang, V. et al. (2010) Pharmacogenomics
and Personalized medicine 3: 145-161
PH-003
Glycosylation of EGFR Extracellular Domain Induces Receptor Stability
Maryam Azimzadeh Irani1,2, Chandra Verma1,2
1Bioinformatics Institute (A*-STAR), Singapore, 2School of Biological Sciences, Nanyang
Technological University
Background:
The Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase glycoprotein, is involved
in maintaining several cellular processes and is implicated in several cancers when
mutated or over expressed. Receptor activation is dependent on ligand binding and
dimerization of the extracellular domain which leads to dimerization of the intracellular
kinase domain, autophosphorylation and regulation of downstream signaling pathways.
The EGFR extracellular domain is heavily glycosylated with oligosaccharides. Several
experimental studies have suggested different roles for glycosylation on EGFR activation,
dimerization, ligand binding and tyrosine kinase activity.
Objective:
In this study we apply molecular modelling/simulations to study the effects of glycosylation
on EGFR extracellular domain.
Methods
Fig 1.
Fully glycosylated dimeric EGFR.
EGFR Increased stability:
RMSF of the CA atoms during the MD simulations suggest that glycosylation is associated
with dampened motions, suggesting that the glycans stabilize the structure. Subdomain
III is the most stabilized while subdomain I is stabilized largely in the proximity
of the ligand. Both dimer interfaces including the dimerization arm from domain II
and the tip of domain IV fluctuate less upon glycosylation.
Hydrogen bonding; persistent interactions seen for protein-glycan:
In the disaccharide-containing system, we observed three highly occupied hydrogen
bonds between the glycans and domain III and IV of EGFR. Hydrogen bonds of domain
III involve the residue Asp323 in which a sidechain oxygen interacts with oxygen atoms
of the N-acetylglucoseamine linked to Asn328. In domain IV a hydrogen bond is seen
between the Cys 515 backbone amide and the oxygen atom of N-acetylglucosamine linked
to Asn 504. In the oligosaccharide-containing system hydrogen bonds observed between
the glycan attached to Asn 172 and domain II. These hydrogen bonds form between the
Gln193 sidechain oxygen atom and Cys 191 backbone oxygen atom and the Mannose linked
to Asn 172. The reduction in the mobility of these amino acids suggests that hydrogen
bonds impart stability to both the sugars and to the interacting EGFR.
Fig 2.
EGFR-glycans hydrogen bonding.
Fig 3.
CA atoms fluctuations of glycosylated and non-glycosylated EGFR.
Packing interactions of Glycans Attached to Asn 151 and Asn 328:
An interesting feature is seen in our simulations. The glycans attached to Asn 151
and Asn 328 move towards and interact with each other, forming one unit. In this process,
the glycan attached to Asn 151 moves towards EGF and the ligand binding site and makes
frequent contacts with Asn 91 from subdomain I and Lys 322 from subdomain III. The
glycan attached to Asn 328 forms the scaffold of this arrangement by making several
contacts with Glu 320, Ser 324, Leu 325, Ser 326 and Thr 330 from subdomain III and
Asn 91 and Ser 92 from subdomain I. Electrostatic potential and per-residue free energy
decomposition calculations from our MD simulations suggest that favorable electrostatic
interactions appear to modulate this interaction. Hence this novel conformational
rearrangement leads to the stability of the structure of domains I and III and appears
to associate in maintaining the EGF binding pocket.
Fig 4.
A) Conformational arrangement of Glycans attached to Asn 151 and Asn 328. B) Electrostatic
contribution of the monoosaccharide residues from the two interacting glycans.
Conclusions:
- Increased stability of glycosylated EGFR arises from hydrogen bond Interactions
between subdomains II, III and IV and the attached glycans.
- Packing of Asn 151 and Asn 328 attached oligosaccharides stabilizes the ligand binding
pocket by a network of Intermolecular contacts and favorable electrostatic interactions
between the two glycans and ligand-binding subdomains of EGFR.
- Stabilization of the ligand-binding subdomains under the influe nce of glycosylation
can elongate the half life of the dimeric active state of EGFR.
PH-004
Dynamic protein complexes mediate reactivity and specificity of complement-like immunity
in Anopheles gambiae
Richard Baxter1
1Yale University Dept. of Chemistry, Dept. of Molecular Biophysics & Biochemistry
Insects possess a complement-like immune response utilizing thioester-containing proteins,
or TEPs. The only arthropod TEP of known structure is Anopheles gambiae TEP1, which
is a key component in the natural immunity of this mosquito to malaria parasites (genus
Plasmodium). Unlike vertebrate complement factors, AgTEP1 does not contain an anaphylatoxin
domain which acts to regulate a massive conformational change accompanying activation
of the protein. The mechanism of AgTEP1 must therefore involve an alternative mechanism
for allosteric regulation of thioester activation. In place of a small internal domain,
a large, heterodimeric complex of two leucine-rich repeat (LRR) proteins, LRIM1 and
APL1C, have been shown to specifically bind and stabilize the active conformation
of AgTEP1. I will present my group’s most recent work in this area. We have shown
that different alleles of TEP1, which are known to influence the vectoral capacity
of wild mosquitoes, differ significantly in their susceptibility to thioester hydrolysis.
Allelic variation is centered on residues at the protein-protein interface within
TEP1 containing the thioester bond. The LRIM1/APL1C heterodimer is shown to form an
extended and flexible ensemble in solution. Two closely-related genes to APL1C, APL1A
and APL1B, can also form a complex with LRIM1, and APL1B LRR domain can form a homodimer.
We propose that a flexible and heterogeneous group ensemble of LRIM1/APL1 dimers interact
with the active conformation of TEP1, thereby producing an array of immune complexes
to protect mosquitoes from a diverse set of pathogens.
PH-005
Conformational Changes of the Ribose ABC Transporter Studied by EPR Spectroscopy
Satchal Erramilli1, Michael Simon2, Matthew Clifton3, Cynthia Stauffacher1
1Purdue University, 2Washington University At St. Louis, 3Beryllium
Bacterial ATP-Binding Cassette (ABC) transporters are vital for nutrient uptake. Structural
and functional studies have identified two distinct types (I and II) of importers
based primarily upon mechanistic differences. The ribose transporter in E. coli is
a tripartite ABC importer consisting of a cytoplasmic ATP-binding cassette protein
with dual fused nucleotide-binding domains (NBD), a transmembrane domain (TMD) homodimer,
RbsC, and a periplasmic substrate binding protein (SBP), RbsB. Results from this study
demonstrate that the ribose transporter shares structural and functional features
with both type I and type II systems. ATP hydrolysis in the NBD is stimulated by the
presence of substrate-loaded SBP, typical of type I systems. Paradoxically, substrate
is released upon association of ribose-loaded-RbsB and RbsC, as revealed by equilibrium
dialysis experiments. EPR experiments demonstrate RbsB samples open and closed conformations
when bound to RbsC, suggesting a pathway for substrate release. The presence of additional
ribose, in turn, has a destabilizing effect on the RbsB-RbsC interaction, a type II-like
behavior. ATP-bound RbsA then associates with the RbsBC complex, with hydrolysis subsequently
leading to SBP dissociation. RbsC promotes a closed conformation of RbsA upon association,
promoting ATP hydrolysis. Quantitative measurements from EPR experiments show hydrolysis
then results in the open conformation of RbsB and destabilizes its interaction with
RbsC. RbsA then dissociates from RbsC following ADP release, atypical for ABC transporters
of either type. Co-purification studies and EPR experiments show that both the nucleotide
and magnesium are required to maintain the complex interactions of RbsC and RbsA.
Finally, transport is accomplished by hydrolysis at a single consensus site, with
the second site rendered degenerate by mutations of conserved amino acids; such functional
asymmetry is not uncommon in ABC exporters, but is heretofore unobserved in importers.
Taken together, the ribose transporter appears to function by a distinct mechanism,
and offers an opportunity to gain insight into non-canonical systems.
PH-006
The Catalytic Cycle of hFEN1 Requires Protein and DNA Conformational Changes, but
Are They Rate-Limiting?
L. David Finger1, Ian A. Bennet1, Andrea Hounslow2, Jack C. Exell1, Nicola J. Baxter2,
Jon P. Waltho3, Jane A. Grasby1
1Centre for Chemical Biology, Department of Chemistry, University of Sheffield, 2Molecular
Biology and Biotechnology, University of Sheffield, 3Manchester Institute of Biotechnology,
University of Manchester
Human Flap Endonuclease-1 (hFEN1) is an essential metallo-nuclease involved in Okazaki
Fragment maturation and long-patch base excision repair. During these processes, bifurcated
nucleic acid intermediates with ssDNA 5’-flaps are generated by polymerase strand
displacement synthesis and then cleaved one nucleotide into the downstream duplex
by FEN1 to create a nicked-DNA that is a suitable substrate for ligase. Until recently,
how hFEN1 achieves tremendous catalytic power (rate enhancements >10exp17) and exquisite
selectivity for the scissile phosphate had been understood poorly (1). In 2011, the
Grasby and Tainer labs solved the structures of hFEN1 in complex with product and
substrate. This study revealed that scissile phosphate selectivity is largely due
to the substrate DNA undergoing a novel Di-Nucleotide Unpairing (DNU), which places
the scissile phosphate diester in contact with the requisite divalent metal ions.
In addition, by comparing the structures of hFEN1 alone (2) and in complex with substrate
and product DNAs (3), Grasby and Tainer proposed a model, whereby protein conformational
changes occur upon binding substrate resulting in placement of key basic residues
that position and/or electrophillically catalyse hydrolysis of the scissile phosphate
diester. Further work using a CD-based assay showed that metals are absolutely required
for DNU, whereas the key basic residues in the active site are not. Surprisingly,
perturbations to the protein structure that are much more distant from the FEN1 active
site (i.e., helical cap) prevent DNA unpairing, implying that the FEN1 protein actively
participates in the unpairing process (4,5); however, how it does remains a mystery.
The maximal multiple turnover rate of hFEN1 reaction is rate-limited by enzyme product
release, whereas hFEN1 kinetics under substrate-limiting conditions ([E]<[S]<Km) suggest
that the enzyme is approaching catalytic perfection (i.e., almost diffusion controlled
- 10exp7 M-1s-1) (6). Previous work has shown that a paralogue of hFEN1 (i.e., T5
FEN1) is not rate-limited by chemistry under single turnover (ST) conditions, but
rather some physical limitation such as conformational change (6). To ascertain whether
the protein and/or DNA conformational changes of hFEN1 mentioned above are rate limiting
under ST conditions, we have initiated kinetic and NMR studies to determine the role
of conformational dynamics in the catalytic cycle of hFEN1.
References:
1. Grasby, J.A. et al. (2011) Trends Biochem. Sci. 37, 74-84.
2. Sakarai, S. et al. (2005) EMBO J. 24, 683-693.
3. Tsutakawa, S.E. et al. (2011) Cell 145, 198-211.
4. Finger, L.D. et al. (2013) Nucl.Acids Res., 41, 9839-9847.
5. Nikesh Patel et al. (2013) J. Biol. Chem., 288, 34239-34248.
6. Finger, L.D. et al. (2012) Subcell. Biochem. 62, 301-326.
PH-007
Dynamical structure changes in binding of pharmaceutical target proteins
Hideaki Fujitani1
1Research Center for Advanced Science and Technology, The University of Tokyo
Owing to the latest advance in the computational technologies of microprocessor, high-speed
inter-processor connection, and parallelization algorithm, all-atom molecular dynamics
(MD) simulations of microsecond time scale are getting popular to study the pharmaceutical
target proteins. An antibody binds to its antigen with structure changes. A small
molecule moves around the target protein and finds an entrance to get into the binding
site. These phenomena can be observed in microsecond simulations, but the vital issue
is whether the adopted force field is enough accurate to correctly describe the phenomena.
We are developing a force field (FUJI) based on general AMBER atom types and AMBER94
van der Waals parameters in order to describe arbitrary organic molecules in a unified
manner including proteins and nucleic acids. We use the first principle theoretical
method to refine the force field in contrast to common empirical fittings to experimental
data. The dihedral torsion parameters of protein backbone were determined to agree
with the torsion energy profiles calculated by high-level quantum mechanical theory
for the model systems of protein backbone. Conformational preferences of dipeptides
in water were measured by vibrational spectroscopy. Comparing with the experimental
distribution of Ramachandran angles of dipeptides, how accurately molecular calculations
predict the conformation distribution of dipeptides were investigated for various
force fields and semiempirical quantum methods. FUJI force field gave the best prediction
score among the various methods (Tzanov et al, 2014). In this work we examine dynamical
structure behaviors of pharmaceutical target proteins by microsecond MD simulations
with FUJI force field. Epiregulin (EPR) is a transmembrane protein with 140 residues
belonging to the epidermal growth factor family. Mature EPR (46 residues) binds to
epidermal growth factor receptor (EGFR), which stimulates the proliferative signaling
in cancer cells. Our EPR antibody has a proline at the residue 103 in the third complementarity-determining
region (CDR) of the heavy chain, which has cis peptide bond in free state and trans
one in the bound state with EPR according to X-ray crystallography analysis. In order
to clarify the structure changes in binding we performed extensive MD simulations
of our antibody (fab; 436 residues) and EPR. The total simulation time was about 240
microseconds. We also performed MD simulations for a protein kinase and a small molecule.
We show how the complex system reaches thermal equilibrium states in simulations from
the stiff X-ray crystal structure. The calculations are compared with experiments
such as X-ray crystal structures and thermodynamic quantities measured by isothermal
titration calorimetry (ITC) and surface plasmon resonance (SPR).
This research has been supported by MEXT SPIRE Supercomputational Life Science (hp130006,
hp140228) and FIRST Kodama project in Japan.
PH-008
Structure-based recombination of drug resistance enzymes: structural and functional
tolerance to new dynamics in artificially-evolved enzymes
Sophie M.C. Gobeil1,2, Maximillian C.C.J.C. Ebert1,2, Jaeok Park1,4,5, Donald Gagné1,5,6,
Christopher M. Clouthier1,3, Jürgen Pleiss7, Nicolas Doucet1,5,6, Albert M. Berghuis1,4,5,
Joelle N. Pelletier1,2,3
1PROTEO, 2Department of Biochemistry, U. of Montreal, 3Department of Chemistry, U.
of Montreal, 4Department of Biochemistry and Department of Microbiology and Immunology,
McGill, 5GRASP, 6INRS–Institut Armand-Frappier, U. du Quebec, 7Institute of Technical
Biochemistry, University Stuttgart
Our understanding of the contribution of protein dynamics to function is still emergent.
In a protein engineering context, do we need to take into account the dynamics in
order to maximize the fitness and function of the resulting proteins? Using high resolution
crystal structures, NMR relaxation dispersion and µs molecular dynamics simulations,
we compare two naturally evolved homologous class A β-lactamases, TEM-1 and PSE-4
which share a high degree of structural and functional conservation. We observed a
conservation of dynamics on a catalytically relevant timescale. This is consistent
with dynamics being an evolutionarily conserved feature. However, laboratory-engineered
chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic
differences, despite the function and structure being conserved. The laboratory-engineered
chimeras are thus functionally and structurally tolerant to modified dynamics on the
timescale of the catalytic turnover. This tolerance of β-lactamases to dynamic changes
could be linked to the high fitness of the naturally evolved proteins and implies
that maintenance of native-like protein dynamics may not be essential when engineering
functional proteins.
PH-009
Conformations of the RNA polymerase clamp throughout the transcription cycle studied
by single-molecule FRET
Sarah Sarah1, Andreas Gietl1, Philip Tinnefeld1, Finn Werner2, Dina Grohmann1,3
1Physikalische Chemie - NanoBioSciences, Technische Universität Braunschweig, 2Institute
of Structural and Molecular Biology, University College London, 3Institute of Microbiology
- Single-Molecule Biochemistry, University Regensburg
Transcription is one of the most fundamental processes in biology and every living
organism depends on it. RNA polymerases (RNAP) are at the heart of the transcriptional
machinery catalyzing the synthesis of RNA in a DNA-dependent fashion. This intrinsically
dynamic process requires the highly coordinated interplay of the RNAP with multiple
nucleic acids and transcription factors through the initiation, elongation and termination
phase of transcription in order to coordinate the movement of the RNAP along the DNA
template and the catalytic action of the enzyme. Conformational rearrangements are
a central theme in the process of transcription and the clamp domain is the main mobile
element of RNA polymerases. Even though structures of eukaryotic RNAPs are available,
it has been extremely difficult to decipher the conformational space sampled by the
RNAP clamp at different stages of transcription. Combining the highly tractable recombinant
archaeal transcription system with single-molecule fluorescence resonance energy transfer
(FRET) measurements allowed us to resolve long-standing questions about the conformational
state of the clamp of archaeal-eukaryotic RNAPs in solution. An immense advantage
of our recombinant system is the possibility to perturb it by introducing unnatural
amino acids for subsequent labelling with fluorescent dyes or to omit subunits from
the multisubunit RNAP. Comparing free, nucleic acid- and factor-bound RNAP complexes
we found that the position of the clamp is modulated during the transcription cycle
in a fashion exceeding the simplified closed-open picture drawn from crystallographic
studies. We show that the clamp adopts at least two distinct conformations in both
initiation and elongation complexes. Moreover, we were able to link conformational
states to the catalytic activity of the RNAP. Transcription factors TFE and Spt4/5,
which both bind to the RNAP clamp domain and stimulate the activity of the enzyme,
shift the equilibria towards one of the main conformations suggesting that both prompt
an allosteric switch that influences the structure of the RNAP. Hence, our data provide
a mechanistic rationale for the function of these factors and provide new insights
into the complex dynamic behaviour of the transcriptional machinery.
PH-010
Solvent models for protein simulations – the good, the bad and the applications
Duy Hua1, Amitava Roy1, He Huang1, Carol Post1
1Purdue University
The solvent environment plays a crucial role in determining the structure, dynamics
and function of a biomolecule. In order to utilize molecular dynamics (MD) simulations
to examine the structural changes of biomolecules, it is imperative that the solvent
environment be accurately modeled. Implicit solvent models (ISMs), in which water
molecules are absent and the solvent effect is estimated using an energy function,
offer a low-cost approach to describe the solvent environment in MD simulations. ISMs
have been used for a variety of biological studies; yet, the performance of implicit
solvation methods in capturing the structural characteristics of proteins has not
been rigorously demonstrated. In this work, three ISMs, namely GBMV II, FACTS, and
SCPISM, were evaluated for their abilities to emulate the solvent environment and
its effect on the structures, dynamics and electrostatic interactions of the Src SH2
domain and the Lyn kinase domain. Structural properties such as phi-psi distribution,
average positional fluctuations, ion pair distance distribution, and all-against-all
rms deviation were used to compare the performance of ISMs to the TIP3P explicit solvent
model. Our study shows that the Src SH2 domains solvated with TIP3P, GBMVII and FACTS
sample similar secondary structures as well as global conformations. Additionally,
GBMVII and FACTS perform relatively well in modeling solvent-accessible electrostatic
interactions that would typically require the presence of water molecules. These charge-charge
interactions, however, are not adequately described in the SCPISM solvent model. Overall,
FACTS is computationally efficient and is a reliable alternative solvent model for
the studies of globular proteins. On the other hand, for non-globular proteins with
complex structures, FACTS does not accurately describe the solvent effect on the sampling
of local interactions and global conformations due to the possibilities of water-mediated
interactions. Our assessment of ISMs in terms of structural features in folded proteins
expands previous studies that have utilized hydration energy as the metric for comparison.
Our work reveals that ISMs show poor performance for non-sphrerical, multi-lobal proteins
and the best use of ISMs remains limited to small, globular proteins.
PH-011
Dissection of the water cavity of yeast Thioredoxin 1: the effect of a hydrophobic
residue in the cavity
Anwar Iqbal1, Fabio C. L. Almeida1, Catarina Miyomoto2, Ana P Valente3, Francisco
Gomes Neto4
1National Center of Nuclear Magnetic Ressonance, Insti of Medical Biochemistry, 2Faculdades
Integradas de Três Lagoas-AEMS, 3Center of Structural Biology and Bioimaging (CENABIO),
UFRJ, 4Laboratory of Toxicology, Instituto Oswaldo Cruz, Fiocruz
Thioredoxins (Trx) are ubiquitous proteins that play a key role in redox state regulation
of the cell. They interact with multiple targets in the cell. Our group solved the
structure of S. cerevisiae (yTrx1) and measured its dynamics in different timescales
(pico to seconds). It is well established that the Asp24 is the proton acceptor in
the reduction process of the target protein. We proposed that Asp24 also works to
couple hydration and motion of the interacting loops. We studied conformational motions
of the residues of water cavity and interacting loops. The water molecules in water
cavity of the protein play a vital role in the redox activity of thioredoxin. The
degree of mutational frustration explains in a great deal the multiple timescales
observed in the protein dynamics in yTrx1 and the mutant D24A. The yTrx1 displays
a complex equilibrium between the ground state and several excited hydration states.
This excited states are more permeable to water and it is key to understand the proton
transfer and activation of Cys33. In this current study, we examined the mutant D24A,
and observed that this small hydrophobic residue induces conformational exchange in
residues exposed to the water cavity, such as Cys33. Along with molecular dynamics
simulations and calculation of mutational frustration levels, we were able to describe
conformational details of the water cavity. It is formed by three independent contiguous
lobes which we called lobes A, B and C. Lobe B is the central lobe that contains the
catalytic important residues Cys33 and Asp24. It closed upon mutation D24A. Lobe A
and C remains open upon mutation, meaning that their hydration are independent. The
NMR structures for the oxidized and reduced state of the Mutant D24A has been studied.
PH-012
In-vitro and in-silico studies of ligand binding to the nuclear receptor PPARgamma
using FRET and MD
Narutoshi Kamiya1, Gert-Jan Bekker1, Takuma Shiraki2, Haruki Nakamura1
1IPR, Osaka University, 2Kinki University
The nuclear receptor PPARgamma is a ligand-dependent transcription factor which regulates
gene expression related to glucose homeostasis and insulin sensitization. It is a
potential therapeutic target for metabolic syndrome, cancer, and inflammatory diseases.
Crystal structures have revealed that its ligands bind deep inside the pocket. However,
how the ligand recognizes PPARgamma and penetrates inside the pocket remains unclear.
We have measured the binding kinetics using the FRET method and have calculated potential
dissociation paths via molecular dynamics (MD) simulations. The binding kinetics were
measured using FRET between the ligand, C8-BODIPY, which is a fluorescent pigment
structurally similar to one of its agonists 15-oxo-eicosatetraenoic acid, and cyan
fluorescent protein (CFP) which is fused to PPARgamma. Fluorescence of C8-BODIPY is
observed when it is close to CFP when CFP is excited by light at 410nm. The ligand
was simultaneously added to the PPARgamma-CFP solution using the stopped-flow method.
Time-resolved fluorescence signals of C8-BODIPY and CFP were also monitored. Two time
constants were observed by the FRET experiment, which correspond with the landing
and docking processes. We used our new MD program, psygene-G [1], which utilizes the
GPU for acceleration of the non-bonded interactions, such as electrostatic interaction
which uses our original zero-dipole summation method [2, 3]. Previously we were able
to attain similar thermodynamics with respect to the particle mesh Ewald method in
membrane proteins and DNA-water-ion systems [4, 5] and have applied it to the thermodynamic
study of dynein [6]. We have introduced random accelerated MD (RAMD) into the psygene-G
program to predict a ligand’s dissociation path from the initial crystal structure
(PDB ID: 2zk6). In RAMD, an external random-directional constant force is added to
the ligand. We executed 96-RAMD simulations with different random seeds for the initial
pull directions. Twelve RAMD trajectories managed to dissociate within 1 ns, while
the remaining simulations did not manage to disassociate within this time-frame. In
all twelve cases the ligand went past R288, which is located at the surface of PPARgamma.
Interestingly, a somatic mutant (R288H) is related to colon cancer. We concluded that
R288 plays a role of a gate keeper to assist ligand binding.
References:
[1] Mashimo T. et al. J. Chem. Theory Comput. 9, 5599 (2013).
[2] Fukuda I. et al. J. Chem. Phys. 134, 164107 (2011).
[3] Fukuda I. et al. J. Chem. Phys. 140, 194397 (2014).
[4] Kamiya N. et al. Chem. Phys. Lett. 568, 26 (2013).
[5] Arakawa T. et al. PLoS ONE 8, e76606 (2013).
[6] Nishikawa Y. et al. J. Mol. Biol. 426, 3232 (2014).
PH-013
Atomic Insight into kinetic mechanism for sumoylation of UBC9 with substrate motif
(Ψ-K-x-D/E) by molecular dynamic simulation
Mooseok Kang1, Wookyung Yu1, Juhwan Lee1, Iksoo Chang1
1Department of Brain Sciences, Daegu Gyeongbuk Institute of Science and Technolog
SUMOylation is a post-translational modification involved in various cellular processes,
such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein
stability, response to stress, and progression through the cell cycle. SUMOylation
is a conjugation of glysine residue of SUMO protein with lysine residue in target(RanGAP1)
protein through the influence of enzyme E2(Ubc9) and E3(RanBP2). SUMO proteins modify
the function of target protein in the cell. But the kinetic mechanism of SUMOylation
is not well-understand. We traced out the full kinetic process of SUMOylation of the
SUMO1-Ubc9 -RanGAP1-RanBP2 complex in atomic detail via molecular dynamic simulation.
We uncovered the kinetics of SUMOylation mechanism and verified the important residues
which play a key role in the SUMOylation process. The comparison of our results with
the experimental one are also discussed.
PH-014
Bending over Backwards for Water: How KCNE3 Helix Curvature and Flexibility Influence
a Human Potassium Channel’s Conduction Profile
Brett Kroncke1,2, Wade Van Horn0, Jarrod Smith1,2, David Nannemann2,6, Jens Meiler2,6,
Carlos Vanoye7, Charles Sanders1,2
1Department of Biochemistry, Vanderbilt University, 2Center for Structural Biology,
Vanderbilt University, 3Department of Chemistry and Biochemistry, Arizona State University,
4Biodesign Institute, Arizona State University, 5Center for Personalized Diagnostics,
Arizona State University, 6Department of Chemistry, Vanderbilt University, 7Northwestern
University Feinberg School of Medicine
Human potassium channel KCNQ1 is expressed in several tissues including inner ear,
heart muscle, lung, intestine, and stomach, each requiring a unique current profile
for proper function. To tune its output current, KCNQ1 complexes with several accessory
proteins from the KCNE family, each KCNE family member modulating KCNQ1 distinctly:
KCNE1 causes the channel to delay opening and become more conductive in the open state,
KCNE3 causes the channel to be constitutively conductive, and KCNE4 closes the channel.
The purpose of this study is to uncover the atomic-scale, mechanical mechanism of
how KCNE3 modulates KCNQ1. To do this we modeled the interaction between KCNQ1 and
KCNE3 with a hybrid experimental-computational approach. Our strategy is to determine
the NMR structure of KCNE3 alone in bilayer-mimicking bicelles, build a homology model
of the open-state KCNQ1, and dock the structure of KCNE3 onto the model of KCNQ1 using
electrophysiology-based restraints to validate and refine the resulting model complexes.
Paramagnetic Relaxation enhancement, residual dipolar coupling and sequence analysis
suggest the single span transmembrane helix of KCNE3 is significantly curved. Hydrogen-deuterium
exchange, nuclear Overhauser effect cross peaks, and AMBER simulation suggest KCNE3
carries a significant amount of water in the C-terminal side of the transmembrane
helix. The 1H-15N two dimensional nuclear magnetic resonance spectra of a known phenotype-changing
mutation in the center of the transmembrane helix, V72T, suggests the helix adopts
multiple conformations when the extra side-chain hydroxyl group is present. A docking
funnel corroborates a binding pocket suggested by the in vivo electrophysiology data
where KCNE3 wedges between a helix near the potassium pore and two helices in the
voltage sensitive element of the channel. Within this pocket, we believe the conformational
sampling and structural rigidity—or flexibility, in the context of the V72T mutant—of
the accessory protein KCNE3 directly influence the modulation profile.
PH-015
Watching conformational changes in proteins by molecular dynamics simulations
Kresten Lindorff-Larsen1
1Department of Biology, University of Copenhagen
Proteins are dynamical molecules and their ability to adopt alternative conformations
is central to their biological function. Examples include motions that underlie allosteric
regulation or ligand binding, or protein dynamics in enzymes that can modulate the
overall catalytic efficiency. Protein motions can often be described as an exchange
between a dominant, ground state structure and one or more minor states. The structural
and biophysical properties of these transiently and sparsely populated states are,
however, difficult to study, and an atomic-level description of those states is challenging.
In an attempt to determine how well molecular dynamics simulations can capture slow,
conformational changes in protein molecules we have studied two different protein
systems which are known to undergo conformational exchange on the millisecond timescale,
and for which structural information is available for both major and minor states.
Using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by
structural information from X-ray crystallography and NMR, we show that current force
fields and sampling methods allow us to sample experimentally-determined alternative
conformations with surprisingly high accuracy. In particular, we find that we can
reversible sample both the ground state and minor state, and that the simulations
capture the structure of the minor states also. Our simulations enable us to calculate
the conformational free energy between the two states, and comparison with relaxation
dispersion NMR experiments demonstrates a high accuracy. Thus, we show for two distinct
proteins that we can map the free energy landscapes and that both the structure and
energetics are in excellent agreement with NMR experiments. Our simulations provide
insight into the structural and biophysical properties of transiently populated minor
states, and help reinterpret previous experimental measurements. Further, our results
demonstrate that, at least in the two cases we have studied, modern simulation methods
enable us to examine these otherwise “invisible” states of proteins and describe their
structural, functional and thermodynamic properties. Our results in this way help
demonstrate how simulations can now add to our knowledge of transiently formed, “hidden”
states of proteins.
PH-016
Coupling Conformational and Energetic Changes in G Protein Signaling
Alyssa Lokits1, Julia Koehler Leman2, Kristina Kitko1,3, Natha Alexander4, Heidi Hamm1,5,
Jens Meiler1,5,6
1Neuroscience, Vanderbilt University Medical Center, 2Chemical and Biomolecular Engineering,
Johns Hopkins University, 3Engineering, Vanderbilt University Medical Center, 4Pharmacology,
Case Western Reserve University, 5Pharmacology, Vanderbilt University Medical Center,
6Chemistry, Vanderbilt University Medical Center
Cell signaling is a fundamental process for all living organisms. G protein-coupled
receptors (GPCRs) are a large and diverse group of transmembrane receptors which convert
extracellular signals into intracellular responses primarily via coupling to heterotrimeric
G proteins. In order to integrate the range of very diverse extracellular signals
into a message the cell can recognize and respond to, conformational changes occur
that rewire the interactions between the receptor and heterotrimer in a specific and
coordinated manner. By interrogating the energetics of these interactions within the
individual proteins and across protein-protein interfaces, a communication network
between amino acids involved in conformational changes for signaling, is created.
To construct this mapping of pairwise interaction energies in silico, we analyzed
Rhodopsin GPCR coupled to a Gαi1β1γ1 heterotrimer. The structure of this G protein
complex was modeled in the receptor-bound and unbound heterotrimeric states as well
as the activated, monomeric Gα(GTP) state. From these tertiary structural models,
we computed the average pairwise residue-residue interactions and interface energies
across ten models of each state using the ROSETTA modeling software suite. Here we
disseminate a comprehensive analysis of all critical interactions and create intra-protein
network communication maps. These networks represent nodes of interaction necessary
for G protein activation.
PH-017
Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA
in complex with the promoter DNA
Yuan-Chao Lou1, Yi-Fen Kao1, Tsi-Hsuan Weng2, Yi-Chuan Li2, Chwan-Deng Hsiao2, Chinpan
Chen1
1Institute of Biomedical Sciences, Academia Sinica, 2Institute of Molecular Biology,
Academia Sinica
PmrA, an OmpR/PhoB family response regulator (RR), takes part in the two-component
system that manages genes for polymyxin resistance through a phosphorylation-dependent
regulation. Phosphorylation of OmpR/PhoB RR induces the formation of a two-fold symmetric
dimer in the N-terminal receive domain (REC), promoting 2 C-terminal DNA-binding domains
(DBDs) to recognize tandem repeat DNA sequences on the promoter to elicit adaptive
responses. Recently, Narayanan et al. presented the complex structure of active-like
KdpE in complex with DNA, which reveals a unique asymmetric REC-DBD interface that
is necessary to form stable complexes for transcription activation. In this study,
we report the 3.2 Å resolution crystal structure of BeF3–activated PmrA in complex
with promoter DNA as well as its dynamics in solution by NMR. Unlike the case of KdpE,
PmrA-DNA complex structure reveals a different asymmetric REC-DBD interaction. Interestingly,
NMR assignments and dynamics study suggest that REC and DBD do not form stable contacts
in solution; instead, 2 domains tumble separately in the absence or presence of DNA.
Relaxation dispersion experiments on methyl groups further show that several REC-DBD
interfacial residues exhibit similar slow dynamics in the presence of DNA. It is hence
highly possible that the slow dynamics observed on these interfacial methyl groups
are related to the formation of asymmetric REC-DBD interaction, which reduces the
mobility of PmrA-DNA complex and promotes crystallization. In solution, two domains
tumble independently and have diverse orientations, which together with the DBD-DBD
interface may facilitate searching best interactions with RNA polymerase holoenzyme
for transcription activation.
PH-018
Time-resolved X-ray Observations of Nano-scale Protein Assembly Networks
Yufuku Matsushita1, Hiroshi Sekiguchi2, Noboru Ohta2, Keigo Ikezaki1, Yuji Goto3,
Yuji Sasaki1,3
1The University of Tokyo, Graduate School of Frontier Science, Advanced Materials
Science, 2SPring-8, 3Osaka University, Institute For Protein Research
Protein aggregation and network formation in diverse protein species had been studying
in various experimental approaches such as absorbance spectrophotometry, dynamical
light scattering and X-ray scattering. However, these conventional methods are only
able to observe averaged information on bulk conditions and also these techniques
have a limitation of time resolved observations. Recently, development of single molecular
observation techniques are remarkable. Diffracted X-ray Tracking (DXT) is one of the
notable single molecular observation methods to capture detailed intramolecular dynamics
of an individual single protein molecule by the labeling X-ray method. In this study,
we present an innovative method for observing the protein assembly networks of nano-scale
size on the bulk solution using a DXT that possess pico-meter scale positional accuracy
and micro-second time resolution. This method is founded by detecting angular rotational
displacement of a coexisted and dispersed single gold nanocrystal (approximately 100
nm) on target solution. For target sample, we choose a crystal precursor metastable
state of 20 mg/ml lysozyme solution (Hen egg white lysozyme: 14331.20 g/mole on 0.2
M sodium acetate 3 – 4 w/v NaCl, pH 4.7 buffer) and 10 mg/ml of lysozyme solution
such as stable condition. For reference sample, we prepare 20 mg/ml and 10 mg/ml of
ribonuclease A solution (Ribonuclease A (Bovine): 13708.40 g/mol, 0.2 M sodium acetate
3 – 4 w/v NaCl, pH 4.7 buffer). The experiment was carried out at SPring-8 BL40XU.
From DXT analysis, we obtained logarithmic rotational velocity distribution of 20
mg/ml, 10 mg/ml lysozyme solution and 20 mg/ml, 10 mg/ml of Ribonuclease A solution
during 1000 µs and we processed regression analysis of Gaussian fitting in each distribution.
From this result, 10 mg/ml of lysozyme solution (stable) and 20 mg/ml, 10 mg/ml (reference)
have definitely regressed by single peak normal Gaussian distribution that are positional
peaks at 3.66 mrad and 1.4 mrad and 1.2 mrad, respectively. In contrast, 20 mg/ml
of lysozyme solution consisted of double peak logarithmic distribution at 3.42 and
9.54 mrad. This peak separation tendency from DXT are typically observed in a supersaturated
condition of the sodium acetate solution which coexisting nano-scale solute networks
(DXT and Small Angle X-ray Scattering (SAXS) experiment). From this study, DXT measurement
results and detailed analysis process for a protein solution such as crystal precursor
metastable state of lysozyme solution are concerned that this technique is a powerful
tool for observing nano-scale protein network’s existence and its local dynamics in
bulk solution. At the present time, we will demonstrate the detailed analysis and
processing from DXT raw data and the conformity of the results from another experiment
confirmation such as SAXS and Darkfield microscopy. Finally, we present a detailed
protein network model of lysozyme metastable solution from DXT result.
PH-019
Functional implications of co-evolving residue sectors in the Ribonuclease A family
Chitra Narayanan1,2, Kimberly Reynolds3, Rama Ranganathan3, Nicolas Doucet1,2,4
1INRS-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies,
2GRASP, Groupe de Recherche Axé Sur la Structure des Protéines, 3649 Promenade S,
3Green Center for Systems Biology, University of Texas Southwestern Medical cent,
4PROTEO, Québec Network for Research on Protein Function, Structure and Engineeri
Increasing evidence suggests that conformational fluctuations experienced by proteins
play a key role in promoting function. However, the experimentally derived dynamic
behavior of discrete protein systems has yet to provide clear evidence on the evolutionary
conservation of motional events between structural and functional protein homologues.
In this work, we used a statistical coupling analysis (SCA) approach to analyze the
role of co-evolving amino acids in protein function and flexibility. SCA facilitates
the identification of distinct residue sectors and provides an understanding of the
correlation between amino acid co-evolution and biological function. As a model system,
we used the Ribonuclease A (RNase A) family, which includes canonical members that
are structurally similar, but perform diverse activities such as angiogenesis, anti-pathogenicity
and neurotoxicity, while preserving the common ribonucleolytic function. Analysis
of 1922 sequences with SCA 6.0 allowed the determination of five independent components
(ICs) corresponding to amino-acid sectors with distinct functional, structural and/or
dynamical roles within the family. While most ICs correspond to residues critical
for protein stability and catalytic function, IC4 correlates with residues experimentally
determined to be highly dynamic on the catalytic timescale in several RNase A homologues,
as we confirmed by NMR 15N-CPMG experiments. Additionally, IC5 residues form a hydrogen-bonding
network shown to allosterically modulate and coordinate catalytically productive motions.
These results provide a direct observation between the role of residue sectors in
stability and motions relevant to function in the RNase A family. This study highlights
the role of concerted action of multiple residues towards a common function, which
can guide experiments for engineering proteins to enhance function such as catalysis.
PH-020
Effects of KCl on the Dynamics and Catalytic Mechanism of a Halophilic Enzyme - Dihydrofolate
Reductase (hvDHFR) from Haloferax volcanii
Sivanandam V. N.1, Ana Laín1, Óscar Millet1
1Structural Biology Unit, CICbioGUNE
Halophilic archea are a type of extremophiles that thrive in hypersaline conditions.
In order to avoid the osmotic shock, their cytoplasm is maintained with higher ionic
strength up to 4-6M. Such a high ionic strength of intracellular environment protects
the cells from desiccation or salting out. However, one wonders how such high salt
concentrations keep the cell functional and active throughout all the cellular reactions
(i.e. enzyme catalysis). The answer lies in the evolution of the halophiles that has
left the halophilic proteins with a certain preference for amino acids composition
[1] (prefers more acidic residues and overall decrease in hydrophobic side-chains).
It’s also known that the salt concentration can affect the activity and the underlying
mechanism [2] of the halophilic enzymes. In order to further understand the haloadaptation,
we have chosen to study a model system, dihydrofolate reductase (DHFR) from Haloferax
volcanii (hvDHFR). DHFR is a very well understood enzyme and known to bound variety
of ligands that can modulate the dynamics and the energy landscapes of the enzyme
[3]. We have probed the μs-ms dynamics of hvDHFR bound to the cofactor NADPH (holoenzyme)
and other substrates (DHF, THF) using relaxation dispersion NMR. In order to understand
the salt contribution to the dynamics and catalytic mechanism, the experiments were
done in the increasing order of salt concentration, shedding light on the conformational
ensembles involved in the catalytic mechanism. The NMR relaxation dispersion analysis
of different enzyme complexes in the presence of 3M and 2M KCl has shown more conformational
fluctuations than compared to the enzyme with 1M KCl. This result suggests that the
enzyme preferably is highly active & samples more flexible conformers in the hypersaline
environment. Further, quantification of these conformers/invisible-states and their
salt-dependence will provide new insights into the mechanism of the enzyme-catalysis.
Also, it will facilitate designing novel enzymes that can be used in the bioprocess
industrial set-up under extreme conditions.
1. Tadeo, X.; López-Méndez, B.; Trigueros, T.; Laín, A.; Castaño, D.; Millet, O. PLoS
Biol 2009, 7, e1000257.
2. Ortega, G.; Lain, A.; Tadeo, X.; Lopez-Mendez, B.; Castano, D.; Millet, O. Sci.
Rep. 2011, 1
3. Boehr, D. D.; McElheny, D.; Dyson, H. J.; Wright, P. E. Science 2006, 313, 1638.
PH-021
Structure and dynamics of the octameric iron-, heme- and cobalamin-binding protein
HbpS from the soil bacterium Streptomyces reticuli
Dario Ortiz De Orue Lucana1, Matthew Groves2, Ina Wedderhoff1
1University of Osnabrueck, 2University of Groningen
We have identified the extracellular protein HbpS in the cellulose degrader Streptomyces
reticuli and shown that it specifically binds iron ions, heme and aquo-cobalamin.
Based on 3D crystal structures, structural alignments, sequence comparisons, mutagenesis,
and comparative biochemical investigations, we identified the coordination sites for
iron, heme and aquo-cobalamin, and binding kinetics were elucidated [1,2]. HbpS interacts
with the membrane-embedded sensor kinase SenS. While under non-stressed conditions
HbpS inhibits SenS autophosphorylation, under oxidative-stressing conditions activates
it. SenS in turn phosphorylates the response regulator SenR that activates the transcription
of anti-oxidative genes [3]. We crystallized HbpS and solved its 3D crystal structure
that revealed an octomeric assembly which is required for interaction with SenS [4].
Using mutagenesis, FRET, CD spectroscopy, fluorescence spectroscopy and site-directed
spin labelling combined with pulse electron paramagnetic resonance spectroscopy, we
demonstrated that iron-mediated oxidative stress induces both secondary structure
and overall intrinsic conformational changes within HbpS. We additionally showed that
HbpS is oxidatively modified, leading to the generation of highly reactive carbonyl
groups and tyrosine-tyrosine bonds [5]. We concluded that these molecular events are
responsible for the HbpS-mediated activation of the sensor kinase SenS. This presentation
will focus on the HbpS structure and dynamics within the protein.
References:
[1] Ortiz de Orue Lucana D, Fedosov SN, Wedderhoff I, Che EN, Torda AE (2014) The
extracellular heme-binding protein HbpS from the soil bacterium Streptomyces reticuli
is an aquo-cobalamin binder. J Biol Chem 289:34214-34228
[2] Wedderhoff I, Kursula I, Groves MR, Ortiz de Orué Lucana D (2013) Iron binding
at specific sites within the octameric HbpS protects streptomycetes from iron-mediated
oxidative stress. PLoS One. doi: 10.1371/journal.pone.0071579.
[3] Siedenburg G, Groves MR and Ortiz de Orué Lucana D (2012) Novel Redox-Sensing
Modules: Accessory Proteins- and Nucleic Acids-mediated Signaling. Antioxid Redox
Signal 16: 668-677.
[4] Ortiz de Orué Lucana D, Bogel G, Zou P and Groves MR (2009) The oligomeric assembly
of the novel haem degrading protein HbpS is essential for interaction with its cognate
two-component sensor kinase. J Mol Biol 386: 1108-1122.
[5] Ortiz de Orué Lucana D, Roscher M, Honigmann A, Schwarz J (2010) Iron-mediated
oxidation induces conformational changes within the redox-sensing protein HbpS. J
Biol Chem 285: 28086-28096.
PH-022
A centrosomal protein FOR20 regulates microtubule assembly through a direct interaction
with tubulin
Dulal Panda1, Shalini Srivastava1, Ilina Bareja1
1Indian Institute of Technology Bombay
FOR20 (FOP-related protein of 20 kDa), a centrosomal protein, is conserved across
all ciliated eukaryotes. It has been shown to be involved in ciliogenesis in RPE cells
and in the S-phase progression in HeLa cells. It localizes to the pericentriolar satellite
and at the centrosome. The localization of FOR20 at the centrosome has been found
to be throughout the cell cycle. In Paramecium, FOR20 is involved in docking of the
basal body to the cell membrane and in the assembly of the transition zone. We found
that FOR20 colocalizes with ciliated microtubules in NIH3T3 cells. Since, the localization
of FOR20 to the pericentriolar satellite is dependent on microtubules and the cilium
itself is a microtubule-based structure; we sought to investigate the role of FOR20
in microtubule assembly. The over-expression of GFP-FOR20 in HeLa cells led to the
depolymerization of microtubules while only the GFP expressing HeLa cells showed typical
microtubule network. In addition, the microtubules of GFP-FOR20 expressing HeLa cells
were found to be more sensitive towards nocodazole, a microtubule-depolymerizing agent,
suggesting that FOR20 destabilizes microtubules. The effect of FOR20 on the polymerization
of tubulin was monitored by light scattering, sedimentation assay and electron microscopy.
In vitro, FOR20 inhibited the rate and extent of polymerization of tubulin. For example,
5, 10, 15 and 20 µM FOR20 inhibited the polymerization of purified tubulin by 11,
20, 39 and 52%, respectively. Further, the sedimentation assay suggested that FOR20
inhibited the polymerization of tubulin. In addition, electron microscopic analysis
of the assembly mixture showed that FOR20 inhibited microtubule formation in vitro.
The binding of FOR20 to purified tubulin was monitored by several complimentary techniques.
Using size exclusion chromatography, FOR20 was found to be co-eluted with tubulin
indicating that FOR20 binds to tubulin. Further, the interaction between FOR20 and
tubulin was analyzed by surface plasma resonance technique and the result showed that
FOR20 binds to tubulin with a modest affinity. The data together suggested that FOR20
regulates microtubule assembly through a direct interaction with tubulin.
PH-023
How amide hydrogens exchange in native proteins
Filip Persson1, Bertil Halle1
1Biophysical Chemsitry, Lund University
Amide hydrogen exchange (HX) is widely used in protein biophysics even though our
ignorance about the HX mechanism makes data interpretation imprecise. Notably, the
open exchange-competent conformational state has not been identified. Based on analysis
of an ultra-long molecular dynamics trajectory of the protein BPTI, we propose that
the open (O) states for amides that exchange by subglobal fluctuations are locally
distorted conformations with two water molecules directly coordinated to the N–H group.
The HX protection factors computed from the relative O-state populations agree well
with experiment. The O states of different amides show little or no temporal correlation,
even if adjacent residues unfold cooperatively. The mean residence time of the O state
is ∼100 ps for all examined amides, so the large variation in measured HX rate must
be attributed to the opening frequency. A few amides gain solvent access via tunnels
or pores penetrated by water chains including native internal water molecules, but
most amides access solvent by more local structural distortions. In either case, we
argue that an over-coordinated N–H group is necessary for efficient proton transfer
by Grotthuss-type structural diffusion.
PH-024
Differences in redox reactions with NADP+/H between ferredoxin-NADP+ oxidoreductases
from Bacillus subtilis and Rhodopseudomonas palustris
Daisuke Seo1, Hidehiro Sakurai2, Pierre Sétif3, Takeshi Sakurai1
1Graduate School of Natural Science and Technology, Kanazawa Univ., 2Research Institute
for Photobiological Hydrogen Production, Kanagawa University, 3CEA, iBiTecS
Ferredoxin-NAD(P)+ oxidoreductase (FNR) is a ubiquitous enzyme that catalyze the redox
reaction between soluble small iron-sulfur protein ferredoxin and NADP+/H. FNRs from
photosynthetic green sulfur bacterium Chlorobaculum tepidum (CtFNR), low-GC content
gram-positive bacterium Bacillus subtilis (BsFNR) and purple non-sulfur bacterium
Rhodopseudomonas palustris (RpFNR) are homo-dimeric proteins containing one FAD prosthetic
group per subunit. Crystal structure analyses of Ct- and BsFNRs have revealed their
significant structural homology to NADPH-thioredoxin reductase from Eschelicia coli,
which is distinct from the monomeric FNRs from plastids of higher plants, cyanobacteria,
γ-proteobacteria and putidaredoxin reductase. Previous studies on steady-state reaction
of Bs- and RpFNRs with NADP+/H by diaphorase assay have demonstrated that NADPH oxidation
rates of BsFNR and RpFNR were comparable to those of the FNRs from plastid and cyanobacteria.
But affinities toward NADP+/H and rates for oxidation of NADPH differed significantly
between BsFNR and RpFNR, despite their high amino acid sequence homology. In this
work, we report pre-steady state reactions of BsFNR and RpFNR with NADP+/H by a stopped-flow
spectrophotometry. Mixing oxidized BsFNR with NADPH yielded a rapid formation of charge
transfer complexes (CTCs) followed by a reduction of the enzyme. BsFNR was almost
fully reduced at equilibrium. Mixing photochemically reduced BsFNR with NADP+ also
rapidly provided an absorption spectrum of CTC but followed reoxidation of reduced
BsFNR was very slow. The amount of oxidized BsFNR after equilibrium depended on NADP+
concentration. Kinetic analyses indicated that the rate-determining steps were the
hydride-transfer reactions in both directions and the rate for the forward direction
was much faster than that for the reverse direction. Mixing oxidized RpFNR with NADPH
exhibited a rapid CTCs formation followed by a slower reduction of the enzyme. Increase
in NADPH concentration reduced the observed rate, suggesting redox potential of RpFNR
was similar to that of NADP+/H couple in the presence of excess NADPH. Mixing photochemically
reduced RpFNR with NADP+ rapidly provided CTCs. The decay corresponding to the reoxidation
of reduced RpFNR with NADP+ involved two distinctive kinetic phases, which would be
due to the similarity in hydride transfer rates of forward and reverse directions,
and the presence of excess amount of NADP+. Kinetic analyses indicated that the rate-determining
steps were the hydride-transfer reactions in both directions and the rate for the
forward direction was close to that for the reverse direction. Obtained data suggested
BsFNR and RpFNR are optimized for different direction of the reaction and may play
different physiological roles.
PH-025
The internal dynamics of fibrinogen and its implications for coagulation and adsorption
Stephan Köhler1,2, Friederike Schmid1, Giovanni Settanni1,3
1Institute of Physics, Johannes Gutenberg University Mainz, Germany, 2Graduate School
Materials Science in Mainz, 3Max Planck Graduate Center with the Johannes Gutenberg-University
Mainz
Fibrinogen is a serum multi-chain protein which, when activated, aggregates to form
fibrin, one of the main components of a blood clot. Fibrinolysis controls blood clot
dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific
locations. Although the main biochemical factors involved in fibrin formation and
lysis have been identified, a clear mechanistic picture of how these processes take
place is not available yet. This picture would be instrumental, for example, for the
design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials
with improved biocompatibility. Here, we present extensive molecular dynamics simulations
of the fibrinogen complex which reveal large bending motions centered at a hinge point
in the coiled-coil region of the complex. This feature, likely conserved across vertebrates
according to our analysis, suggests an explanation for the mechanism of exposure to
lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. It also explains
the conformational variability of fibrinogen observed during its adsorption on inorganic
surfaces and it is supposed to play a major role in the determination of the hydrodynamic
properties of fibrinogen. In addition the simulations suggest how the dynamics of
the D region of fibrinogen may contribute to the allosteric regulation of the blood
coagulation cascade through a dynamic coupling between the a- and b-holes, important
for fibrin polymerization, and the integrin binding site P1.
PH-026
Membrane curvature – the assembler of proteins
Mijo Simunovic1,2, Gregory Voth1, Patricia Bassereau2
1Chemistry Department, The University of Chicago, 2Physico-Chimie Curie, Institut
Curie
Many biological phenomena require the membrane to change its shape. This process is
often mediated by curvature-generating proteins, most notably by those containing
one of many BAR domains. At the same time, membrane curvature controls the way proteins
interact with one another and so it acts as a vital signaling mechanism in the cell.
We combine theoretical modeling with microscopy imaging techniques to study the driving
force underlying the reshaping of biological membranes induced by N-BAR proteins.
In particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical
simulation methods to study the assembly of proteins on the membrane at molecular
resolution. This approach allowed us to elucidate the precise mechanism by which cell
membranes rapidly and from large distances recruit proteins to membrane-reshaping
sites. It also let us identify a surprising role of membrane tension in directing
the strength and geometry of protein-protein interactions. By complementing our simulations
with fluorescence and atomic force microscopies, we study the way molecular assembly
of proteins affects the membrane morphology at a more macroscopic level. We demonstrated
how large-scale ordering of the proteins on the membrane is mediated by a strikingly
long-range interaction that is driven by membrane fluctuations. In sum, our combined
theoretical and experimental approach gives vital clues on how the mechanical properties
of the membrane may regulate protein dynamics in living cells.
PH-027
Transmission of rigidity at a distance - new insights into allosteric signalling in
G-Protein Coupled Receptors
Adnan Sljoka1, Alexandr Bezginov2
1Kyoto University, 2University of Toronto
Understanding how a protein functions depends in critical ways on predicting which
parts are rigid and which are flexible. Using rigidity-theoretical techniques, a number
or programs such as FIRST, ProFlex or Kinari can rapidly decompose a protein into
flexible and rigid regions. In this study we extend this technique and develop a novel
computational approach for detecting protein allosteric interactions. It is widely
believed that the binding of a ligand at the allosteric site triggers a conformational
change that is transmitted through the protein to cause a rearrangement and alteration
of the shape of the active site. Allostery was first described more than 50 years
ago; however, the underlying allosteric mechanism is still not well understood. We
will introduce a rigidity-based allosteric mode of communication together with an
algorithm which can detect transmission of rigidity and shape changes between two
(or more) distant binding sites in allosteric proteins. Our algorithm is also used
to predict and identify regions in the protein that are critical for the coupled communication
between distant sites (i.e. allosteric pathways). Starting with a set of known GPCR
3-dimensional structures, we apply our methods and show how binding of an activating
ligand (i.e. agonist) triggers small rigidity changes which propagate to the critical
G-protein binding regions. In contrast, in the inactive GPCR structures no such rigidity
allosteric communication is observed. Detailed predictions and analysis on activated
(agonist-bound) and inactive adenosine receptors is discussed and results are compared
with experimental evidence. These results show that rigidity-based allosteric model
and algorithm is a powerful new tool for detecting allostery in GPCRs.
PH-028
Comparing the intrinsic dynamics of multiple proteins using elastic network models
reveals global similarities based on their overall shape
Sandhya Tiwari1,2, Nathalie Reuter1,2
1Department of Molecular Biology, University of Bergen, 2Computational Biology Unit,
Department of Informatics, University of Bergen
With increasing protein structural data, we find ourselves with the opportunity to
study the dynamical similarities within large ensembles in order to understand the
role of protein dynamics at various levels. Is there evolutionary pressure to conserve
dynamics? Is this necessary to retain functionality, as it has been suggested? We
performed strategic comparative analysis using the elastic network model, which has
proven to be highly efficient and informative (Fuglebakk et al., BMC Bioinformatics,
2014) to investigate how the dynamics of related proteins change when their functional
or oligomeric state shifts, and if it is conserved within protein families. In our
work, we have found that proteins with different ligand-binding states, as in adenylate
kinase (Tiwari et al., BMC Bioinformatics, 2014), or oligomeric states, as in the
PyrR family (Perica et al., Science, 2014), can be classified based on the global
similarity of their intrinsic dynamics. Moreover, our recent analysis on functionally
distinct protein families with the TIM barrel fold indicates that the overall similarity
in shape dictates similarity in intrinsic dynamics regardless of sequence and functional
conservation or evolutionary links. We suggest that since shape dictates a large part
of dynamical similarity in proteins, the changes in local flexibility play a strong
role in differentiating various functional states.
PH-029
Oxygen-Affinity and Cooperativity of Hemoglobin (Hb) are Regulated by 4D Structural
Changes (Protein Dynamics), rather than 3D Structural Changes
Takashi Yonetani1, Kenji Kanaori2
1University of Pennsylvania, Department of Biochemistry & Biophysics, 2Kyoto Institute
of Technology, Department of Bio-Molecular Engineering
Introduction: The widely-held mechanism of allostery of Hb has been that the changes
from the R-quaternary/tertiary structures of oxy-Hb to the T-quaternary/tertiary structures
of deoxy-Hb exert certain constraints on the coordination structure of the heme group,
leading to a lower O2-affinity of the hemes and thus that of Hb [1]. However, we found
that there is no causal correlation between the static T/R-quaternary structures and
the low/high O2-affinities of Hb, respectively [2]. We explore an alternative mechanism
of the regulation of the ligand-affinity and cooperativity in Hb. Results and Discussion:
The O2-affinities of free Fe[II]-protoporphyrin IX-nitrogenous base complexes in organic
solvents are very low (P50 > 102 ∼ 103 Torr), whereas the apparent O2-affinities of
these metalloporphyrins, which are incorporated in apo-myoglobin, apo-Hb, serum albumin,
etc., increase substantially to P50 < 10-1 ∼ 101Torr, though their coordination structures
are apparently unchanged [3]. Such substantial increases in the apparent ligand-affinities
of metalloporphyrin-containing proteins are accomplished by preventing/inteferring
with the dissociation of the ligand by protein matrix, since the interior of globin
is nearly fully packed by protein matrix. In Hb, the dissociation process of the ligand
proceeds through the “caged” state [4-6], which can be produced by cryogenic photolysis
of the ligated-states at 4.2K and in which the metal-ligand bond is broken and the
un-bonded ligand is trapped near the bonding site within the globin moietiy. This
“caged” state has spectral features distinct from those of either deoxy- or ligated
states of the respective hemoproteins. The apparent ligand-affinities of Hb are regulated
by heterotropic effectors without detectable changes in either static quaternary/tertiary
structures of the globin moiety or the coordination/electronic structures of the metalloporphyrin
moiety and thus the ligand-affinity of the metalloporphyrins themselves [7-9]. The
reduction of the apparent ligand-affinities of Hb may be caused by increases in the
migration rate of ligands through globin matrix from the “caged” state to solvent,
resulting from the effector-linked, enhanced high-frequency thermal fluctuations which
increase the transparency of the globin matrix toward small diatomic ligands [7-9].
Conclusion: The ligand-affinity of Hb is regulated through protein dynamics by heterotropic
effectors, rather than static quaternary/tertiary structural changes. Thus, the “caged”
state of Hb acts as a critical transition state in regulation of the affinity for
small diatomic ligands in Hb [9].
[1] M.F. Perutz, Nature 1970 228, 726;
[2] T. Yonetani, et al., JBC 2002 277, 34508;
[3] H. Yamamoto, et al., Bioinorg. Chem. 1977 7, 189;
[4] T. Iizuka, et al., BBA 1974 351, 182;
[5] T. Iizuka, et al., BBA 1974 371, 126;
[6] T. Yonetani, et al., JBC 1974 249, 2168;
[7] T. Yonetani, M. Laberge, BBA 2008 1784, 1146;
[8] M. Laberge, T. Yonetani, Biophys. J. 2008 94, 2737;
[9] T. Yonetani, K. Kanaori, BBA 2013 1834,1873.
PH-030
On the Role of Metal Ions in Synaptic Proteins Assembly
Rafal Jakubowski1, Jakub Rydzewski1, Wieslaw Nowak1
1Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University
The role of metal ions in the regulation of life processes is extremely important.
They act as signal transducers, protein configuration stabilizers, enzymatic cofactors,
oxygen transport supporters and many others. For example, subtle perturbations in
calcium homeostasis may lead to mental disabilities and are linked to diseases such
as Autism Spectrum Disorders (ASD). In this study we focus on complex protein systems,
mainly those present in the brain. We search for dimers mediated by the presence of
metal ions, and determine the impact of the presence or absence of the latter on the
structure and energetic properties of the complex in the protein-protein interface.
We investigate ions’ influence on the interface stability using classic molecular
dynamics methods (MD), including Steered MD. Moreover, we apply a novel suite of enhanced
MD-based methods recently developed by our team (Rydzewski & Nowak) to explore ion
diffusion pathways in protein fragments of the synapses. Finally, we describe specific
inter-protein ion binding motifs with the most important interactions, collating them
with various structures deposited in the Protein Data Bank [1].
This research was supported by “Krok w przyszlosc – 5 edycja” scholarship from the
Marshal of Kuyavian-pomeranian voivodeship (RJ).
[1] H.M. Bermanet al., NAR, 2000, 28, 235-242
PH-031
The role of the Mg(II) ion on integrin-collagen interactions: regulating affinity
through conformational fluctuations
Ana Monica Nunes1,2, Jie Zhu1,2, Jackie Jezioro1,2, Conceição Minetti1, David Remeta1,
Samir Hamaia3, Richard Farndale3, Jean Baum1,2
1Department of Chemistry & Chemical Biology, Rutgers University, 2Center of Integrative
Proteomics Research, Rutgers University, 3Department of Biochemistry, University of
Cambridge
The binding of integrins to collagen plays a critical role in numerous cellular adhesion
processes including platelet activation and aggregation, a key process in clot formation.
Collagen is an unusually shaped ligand, and its mechanism of recognition and role
in selectivity and affinity are unique, and at this stage not well understood. The
I-domain of the integrin protein binds to collagen specifically at multiple sites
with variable affinities, however the molecular mechanism of integrin I-domain (αI)
regulation remains unknown. Using NMR, along with isothermal titration calorimetry,
mutagenesis, and binding assays we are developing a novel integrated picture of the
full recognition process of the integrin α1I binding to collagen. The adhesion of
the α1β1 integrin receptors to collagen is cation-dependent with collagen binding
a Mg(II) ion that is located at the top of the extracellular integrin α1I-domain (α1I).
Our results show evidence for a regulatory effect of the Mg(II) ion on α1I affinity,
by inducing allosteric ms-µs motions of residues distant from the binding site. We
propose a novel model of α1I recognition to collagen, comprising a two-step mechanism:
a conformational selection step, induced by Mg(II) coordination, and an induced-fit
step caused by collagen binding. Hydrogen-deuterium exchange experiments show that
the induced-fit step is facilitated by the reduced local stability of the C-terminus.
We propose that the conformational selection step is the key factor that allows discrimination
between high and low affinity collagen sequences.
PH-032
Effect of Membrane Composition on the Structure of Membrane-Attached Cytochrome P450
3A4
Veronika Navratilova1, Marketa Paloncyova1, Michaela Kajsova1, Karel Berka1, Michal
Otyepka1
1RCPTM, Department of Physical Chemistry, Faculty of Science, Palacky University
Cytochromes P450 (CYP) are heme containing enzymes involved in the metabolism of endobiotics
and xenobiotics, such as drugs or pollutants. [1] In humans, CYPs are attached to
the biological membranes of endoplasmic reticulum or mitochondria by N-terminal transmembrane
anchor and they are partially immersed by their catalytic domain to different level.
[2] Generally, the composition of lipid membrane may significantly affect behavior
of protein embedded in respective membrane e.g. the cholesterol in membrane alters
membrane properties such as: thickening of the membrane, changing the stiffness or
enhancing ordering of the membrane. Furthermore, the increasing amount of cholesterol
in membrane may also alter interaction with membrane proteins and affect solute partitioning
between membrane and water molecules. [3] Cholesterol is also known to noncompetitively
inhibit the most typical drug-metabolizing CYP - CYP3A4, [4] however the mechanism
was unknown. For this reason, we prepared the set of simulations of CYP3A4 embedded
in DOPC lipid bilayers with various cholesterol concentrations (0, 3, 6, 20 and 50%
wt; Figure 1) and the 200ns+ long MD simulations were carried out. MD simulations
showed the formation of funnel-like shape of the lipids close to the catalytic domain
of CYP. In addition, the cholesterol molecules have tendency to accumulate in the
vicinity of membrane-attached F/G loop. The catalytic domain sunk deeper into the
membrane with cholesterol and also the number of amino acids in contact with membrane
was bigger than in the pure DOPC bilayer. In contrast, the presence of higher amount
of cholesterol affected the pattern of channel opening effectively blocking the access
to the active site from the membrane, which in turn may affect the substrate preferences
and catalytic efficiency. [5] Finally, we study the effect of different lipid types
on membrane-attached CYP3A4.
Figure 1:
Difference in orientation and position of CYP3A4 on lipid bilayers with and without
high amount of cholesterol.
1 – Anzenbacher, P., et al.; Cell. Mol. Life. Sci. 2001, 58(5-6):737-47
2 – Berka, K., et al; J. Phys. Chem. B 2013, 117(39): 11556-64
3 – Krause, MR, Acc. Chem. Res. 2014, 47(12) 3512-21
4 – Shinkyo, R., et al.; J. Biol. Chem. 2011, 286: 18426-33
5 – Navrátilová, V., et al.; J. chem. Inf. Model 2015, 55(3): 628-635
Acknowledgement:
IGA_PrF_2015_027, NPU LO1305, GACR P208/12/G016
PH-033
Generation of single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl and
analysis of its structural dynamics
Yusui Sato1, Yusuke Tanaka1, Hiroshi Sekiguchi2, Satomi Inaba1, Takahiro Maruno3,
Yuji C. Sasaki4, Yuji Kobayashi3, Takachika Azuma5, Masayuki Oda1
1Graduate School of Life and Environmental Sciences, Kyoto Prefectural University,
2Japan Synchrotron Radiation Research Institute, 3Graduate School of Engineering,
Osaka University, 4Graduate School of Frontier Sciences, The University of Tokyo,
5Research Institute for Biological Sciences, Tokyo University of Science
Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies are one of the most widely analyzed
type of antibodies, especially with respect to affinity maturation [1-3]. Affinity
maturation is a process in which B cells produce antibodies with increased affinity
for the antigen during the course of an immune response, and is like “evolution” in
term of increasing antigen-binding affinity. During the course of affinity maturation,
the structural dynamics of antibodies, which are closely correlated with the binding
function, can change. To analyze the structural dynamics at atomic resolution and
the single-molecule level, we tried to express and purify single-chain Fv (scFv) antibodies
against NP. Using scFv antibodies, we can also analyze the effects of key residues
on affinity maturation via site-directed mutagenesis. As the first step, we have succeeded
in generating a sufficient quantity and good quality of scFv of affinity-mature anti-NP
antibody, C6, with a linker composed of four repeats of GGGS. The scFv protein was
expressed in the insoluble fraction of E. coli, and solubilized using 8 M urea, followed
by refolding by step-wise dialysis to decrease the urea concentration. The final step
of purification using an antigen column indicated that approximately 2% of the solubilized
protein was correctly refolded and possessed antigen-binding ability. The analytical
ultracentrifugation (AUC) analysis showed that the purified C6 scFv exists in the
monomeric state with little oligomeric contamination. The secondary structure and
thermal stability of C6 scFv were analyzed using circular dichroism (CD). The far-UV
CD spectra of C6 scFv indicated typical β-sheet-rich structures. Upon antigen binding,
the far-UV CD spectrum remained unchanged, but the thermal stability increased by
approximately 20oC. The antigen-binding function of C6 scFv was analyzed using a surface
plasmon resonance (SPR) biosensor, Biacore. The binding affinity and kinetics of C6
scFv for NP conjugated to bovine serum albumin immobilized on the sensor chip were
similar to those of intact C6. Taken together, the results of AUC, CD, and SPR indicated
that C6 scFv could be refolded successfully and would possess its functional structure.
Next, to analyze the structural dynamics of C6 scFv in the absence or presence of
antigen, experiments involving diffracted X-ray tracking (DXT) were performed [4].
C6 scFv with an N-terminal His-tag was immobilized on substrate surfaces using tag
chemistry, and Au-nanocrystals were labeled on the surface of scFv as tracers. The
motions of C6 scFv were analyzed in two rotational directions representing tilting
(θ) and twisting (χ) Mean square displacement (MSD) analysis from more than 200 trajectories
showed that the slope for C6 scFv without antigen, especially in the θ direction,
was greater than that for C6 scFv with antigen, suggesting that the motion of scFv
was suppressed on antigen binding.
[1] Furukawa et al., Immunity 11, 329, 1999.
[2] Sagawa et al., Mol. Immunol. 39, 801, 2003.
[3] Murakami et al., Mol Immunol. 48, 48, 2010.
[4] Sekiguchi et al., Sci. Rep. 4, 6384, 2014.
PH-034
Antibiotic binding drives catalytic activation of aminoglycoside kinase APH(2’’)-Ia
Shane Caldwell1, Albert Berghuis1
1McGill University
The antibiotic resistance enzyme APH(2’’)-Ia confers antimicrobial resistance to aminoglycoside
antibiotics in staphylococci and enterococci. This kinase phosphorylates aminoglycosides
such as gentamicin and kanamycin, chemically inactivating the compounds.
We have determined multiple structures of the enzyme in complex with nucleoside and
aminoglycoside substrates and cofactor magnesium. Introduction of aminoglycoside to
crystals of APH(2’’)-Ia induce gross conformational changes in crystallo, illustrating
several important stages of the catalytic cycle of the enzyme. An interaction between
nucleoside triphosphate and an amino acid residue on a conserved loop has also been
identified that appears to govern a conformational selectivity and modulates the enzyme
activity when no substrate is present.
Comparisons between multiple protein molecules both within and between crystal structures
allow us to infer functional states of the enzyme as it carries out catalysis. These
structures collectively highlight an enzymatic flexibility that not only allows the
binding of diverse aminoglycosides, but also appears to transition from a stabilized,
inactive enzymatic state to a catalytically active enzyme with an active site geometry
identical to distantly-related eukaryotic protein kinases. Mechanistic insight gained
from these studies begin to demystify a widespread staphylococcal resistance factor,
and provide a starting point for the development of anti-infectives toward this important
antimicrobial resistance machine.
PH-035
Disease Related Mutation Effects on Conformations and Dynamics of the Zinc-Finger
NEMO
Ryan Godwin1, William Gmeiner2, Freddie Salsbury1
1Wake Forest University - Department of Physics, 2Wake Forest University Health Sciences
- Department of Cancer Biology
The zinc-finger of the NF-κB Essential Modulator (NEMO) is a ubiquitin binding domain,
and an important regulator of various physiological processes including immune/inflammatory
responses, apoptosis, and oncogenesis. The nominally functioning 28 residue monomer
(2JVX) is represented by a ββα motif, with a CCHC active site coordinating the zinc
ion. Here, we investigate the effects of a single point mutation that has been linked
to the disease states associated with ectodermal dysplasia. The single mutation of
the last binding cysteine (residue 26) to a phenylalanine (2JVY) distorts the available
conformation and dynamics of the protein, as shown via microsecond, GPU-accelerated
Molecular Dynamics simulations. We examine these two proteins in various states of
zinc-binding and coordinating cysteine protonation. In addition to destabilization
of the alpha-helix induced by the cysteine to phenylalanine mutation, prominent conformations
show the β-sheets turned perpendicular to the alpha-helix, providing a possible mechanism
for the induced disease state.
PH-036
Structural characterization of the binding of HIV-1 integrase to its cellular co-factor
Ku70
Ekaterina Knyazhanskaya1, Andrey Anisenko2, Marina Gottikh3, Timofei Zatsepin1
1Moscow State University, Chemistry department, 2Moscow State University, Department
of bioengineering and bioinformatics, 3Moscow State University, Belozersky institute
of physico-chemical biology
Ku is a heterodimer complex composed of two DNA-binding subunits, Ku70 and Ku80, displaying
essential functions for human cell survival. At the same time, Ku has been identified
as a cellular factor important for HIV-1 replication. It is known that the levels
of viral replication are significantly lowered in cells depleted of Ku70. Reportedly,
Ku complex may act at different stages of the HIV-1 cycle, such as the formation of
2-LTR circles, integration and transcription of the integrated provirus. Remarkably,
Ku may also be incorporated into virions during viral propagation. However, the precise
impact of Ku on HIV-1 expression remains unclear and requires further examinations.
A putative role of Ku in the HIV-1 integration is that its Ku70 subunit protects viral
integrase (IN) from degradation by a direct interaction. Thus, the inhibition of Ku70-IN
interaction might affect viral replication. A detailed structure of Ku70/IN complex
would greatly facilitate the inhibitor design. In our work we have proven the existence
of a stable complex between purified Ku70 and HIV-1 IN with a Kd ∼ 70 nM. To gain
insights on the structure of Ku70/IN complex, we performed a systematic analysis of
subdomains within IN that are required for the complex formation. We used both pull-down
and SPR-technique to elucidate interactions of full-length Ku70 with IN fragments.
N-His6-tagged HIV-1 IN separate domains (N-terminal (1-50 aa), catalytic (51-220 aa)
and C-terminal (220-270 aa)) were expressed in E. coli. Several truncated IN variants
containing amino acids 1-160, 1-220, 51-160 and 51-280 were also prepared. A full-size
Ku70 with a GST-tag on its N-terminus was purified from E. coli. All the experiments
performed showed that neither N-terminal nor C-terminal domains of HIV-1 IN are essential
for its binding with Ku70 despite a weak binding capacity retaining to the C-terminal
domain. The catalytic core (51-220 aa) as well as the mutant lacking C-terminal domain
(1-220) both demonstrated affinity to Ku70 comparable to the affinity of the full-size
IN, whereas its truncated variant (51-160 aa) bound to Ku70 protein only weakly. We
also expressed a C-terminal HA-tagged full-length IN and its 1-220 variant in HEK
293T cells together with a WT Ku70-3FLAG and showed that both IN variants are stabilized
by co-expression with Ku70 by approx. twofold. We hypothesize that the binding surface
within IN lies in the region from 160 to 230 a.a. that is a long α-helix. We have
shown that a homologous integrase from prototype foamy virus that lacks this structural
element does not bind to Ku70. It is worth noting that Ku70 does not affect the interaction
of IN with its major cellular partner – LEDGF/p75 as well as its interaction with
the DNA substrate.
This work was supported by an RFBR grant 14-04-00833 and by an RSCF grant 14-14-00489.
PH-037
Structural characterization of calmodulin bound to the intracellular calmodulin binding
domain of Kv7.2 channels by NMR
Ganeko Bernardo Seisdedos1,2, Álvaro Villarroel2, Oscar Millet1
1CIC-Biogune, 2Unidad de Biofísica (CSIC-UPV/EHU)
Mammalian KCNQ genes encode five Kv7 potassium channel subunits (Kv7.1-Kv7.5). Kv7.2
and Kv7.3 are expressed in the nervous system, being the principal molecular components
of the slow voltage gated M-channel, which exert a strong control in neuronal excitability.
Calmodulin (CaM) binds to two sites named helix A and B within the intracellular C-terminus,
mediates inhibition of Kv7.2 channels and is required for the channels to exit the
endoplasmic reticulum. Whereas the understanding of its regulation and its electrophysiological
properties has increased dramatically in the last years, still little is known about
Kv7 potassium channels structure, mainly because obtaining the large amounts of purified
protein required for crystallography or NMR has proven to be very challenging. Here,
we present a structural characterization of the cytosolic domains of Kv7.2, as studied
by NMR spectroscopy. Due to the large size of the protein, we have devised a strategy
where fragments of increasing size have been structurally characterized in complex
with CaM. The dynamic and functional properties of the CaM-Kv7.2 cytosolic domains
are discussed.
PH-038
Cytochrome P450 Oxidoreductase Simulations: Cofactors Movement and Structural Changes
Martin Srejber1, Veronika Navratilova1, Michal Otyepka1, Karel Berka1
1RCPTM, Department of Physical Chemistry, Faculty of Science, Palacky University
The NADPH-dependent Cytochrome P450 Oxidoreductase (CYPOR) is large 677 amino-acid
long microsomal multidomain enzyme responsible for electron donation to its redox
partner cytochrome P450 (CYP) involved in drug metabolism. Electron transfer (ET)
chain is mediated by two riboflavin-based cofactors – flavin mononucleotide (FMN)
and flavin adenine dinucleotide (FAD) within their respective domains and nicotinamide
adenine dinucleotide phosphate (NADPH). During this electron transfer CYPOR undergoes
several structural changes in open and closed state of both domains in different degree
of contact. In spite of the fact that CYP-CYPOR complexes play a key role in drug
metabolism, the atomistic mechanism of structural rearrangements during complex electron
transfers is still lacking. Here, we present the results of our study on structural
changes during CYPOR multidomain complex movement between individual electron transfers
using classical molecular dynamics (MD) and metadynamics (MTD) simulations with cofactors
of NADPH, FAD and FMN in resting state. Homology model of human CYPOR in both forms
(opened and closed) were embedded into pure dioleoylphosphatidylcholine (DOPC) bilayer.
After system equilibration (Figure 1), structural changes of protein, anchor and cofactor
movement were studied. We were able to select possible CYPOR-membrane orientation
which would allow interaction with cytochrome P450. In addition, spontaneous closing
of open CYPOR was observed. However structural changes between crystal structures
and structures obtain from MD simulations lead us to the use of metadynamics in order
to speed up the process. FMN and FAD cofactor remained in close van der Waals contact
during the 100-ns long simulation stabilized by π stack interaction of FAD with Trp676,
whereas continual movement of NADPH continually weakens its π stack interaction with
FAD. After 100 ns of classical MD additional metadynamics simulations were performed
in order to investigate internal motion of cofactors during electron transfer. Atoms
C4N (NADPH) and N5 (FAD) which are responsible for ET were able to move closer to
the distance of 3 Å after adding biasing potential. This distance is more than sufficient
for electron transfer to occur. After switching back to classical MD cofactors got
into resting positions (8 Å) again. Our results show that CYPOR undergo several structural
changes and internal motions of cofactors in order to transfer electrons to its redox
partner - CYP.
Figure 1:
CYPOR equilibrated membrane structure model in closed conformation
Acknowledgement:
IGA_PrF_2015_027, NPU LO1035, GACR P208/12/G016
PH-039
Single Molecule Motion Map of Pentameric Ligand Gated Ion Channel by Diffracted X-ray
Tracking
Hiroshi Sekiguchi1, Yufuku Matsushita2, Yuri Nishino3, Keigo Ikezaki2, Atsuo Miyazawa3,
Tai Kubo4, Christele Huron5, Jean-Pierre Changeux5, Pierre-Jean Corringer5, Yuji Sasaki2
1Research & Utilization Div., JASRI/SPring-8, 2Grad. School Frontier Sci., Univ. Tokyo,
3Grad. Sch. Sci., Univ. Hyogo, Japan, 4National Institute of Advanced Industrial Science
and Technology, Japan, 5Pasteur Institute
Pentameric ligand-gated ion channels (pLGICs) are a major family of membrane receptors
that open to allow ions to pass through the membrane upon binding of specific ligands.
pLGICs are made up of five identical (homopentamers) or homologous (heteropentamers)
subunits surrounding a central pore. Structural information about their multiple allosteric
states, carrying either an open or a closed channel, has become available by recent
studies by X-ray crystallography. However, dynamic information are needed to understand
their mechanism of gating, notably the long-range allosteric coupling between the
agonist binding site and the ion channel gate. Here we used the diffracted X-ray tracking
(DXT) method (1) to detect the motion of the extracellular and transmembrane domain
two pLGICs: the nicotinic acetylcholine receptor (nAChR) and a proton-gated bacterial
ion channel from Gloeobacter called GLIC. DXT is a powerful technique in biological
science for detecting atomic-scale dynamic motion of allosteric proteins at the single
molecular level and at tens of micro seconds timescale resolution. The dynamics of
a single protein can be monitored through trajectory of a Laue spot from a nanocrystal
which is attached to the target protein immobilized on the substrate surface (2,3).
DXT detects two kinds of rotational motions of nanocrystal, tilting and twisting,
based on X-ray incident beam axis. DXT analysis with 0.1ms/f time resolution showed
that tilting motion of the transmembrane domain of GLIC and both tilting and twisting
motions of the extracellular domain of GLIC and nAChR were enhanced upon application
of agonists (lowering the pH for GLIC, and binding of acetylcholine for nAChR). The
detailed dynamic information, including size effect of gold nanocrystal to the motion
of them, is discussed.
[1] Y.C. Sasaki et al., Phys. Rev. E 62:3843 (2000)
[2] H. Sekiguchi et al, PLOS ONE 8:e64176 (2013)
[3] H. Sekiguchi et al, Scientific Reports 4:6384 (2014)
PH-040
Evolutionary hinge migration sheds light on the mechanism of green-to-red photoconversion
in GFP-like proteins
Rebekka M. Wachter1, S. Banu Ozkan2, Hanseong Kim1, Taisong Zhou2
1Department of Chemistry and Biochemistry, Arizona State University, 2Center for Biological
Physics, Department of Physics, Arizona State University
Proteins possess unique structure-encoded dynamics that underlie their biological
functions. Here, we provide experimental evidence for an evolutionary mechanism driven
solely by long-range dynamic motions without significant backbone adjustments, catalytic
group rearrangements, or changes in subunit assembly. Crystallographic structures
were determined for several ancestral GFP-like proteins that were reconstructed based
on posterior sequence predictions, using members of the stony coral suborder Faviina
as a model system. The ancestral proteins belong to the Kaede-type class of GFPs,
a group of proteins that undergoes irreversible green-to-red photoconversion and is
therefore frequently employed in superresolution microscopy. Surprisingly, we find
that the structures of reconstructed common green ancestors and evolved green-to-red
photoconvertible proteins are very similar. Therefore, we analyzed their chain flexibility
using molecular dynamics and perturbation response scanning. We find that the minimal
number of residue replacements both necessary and sufficient to support light-induced
color conversion provide for increased fold stiffness at a region remote from the
active site. At the same time, the allosterically coupled mutational sites appear
to increase active site conformational mobility via epistasis. These data suggest
that during evolution, the locations of fold-anchoring and breathing regions have
been reversed by allosteric means. Therefore, we conclude that the green-to-red photoconvertible
phenotype has arisen from a common green ancestor by migration of a knob-like anchoring
region away from the active site diagonally across the beta-barrel fold. Based on
titration experiments, we estimate that at pH 6, 0.1% of the protein population harbors
neutral side chains for His193 and Glu211, residues that form an internal salt bridge
near the chromophore. We propose that this reverse-protonated subpopulation constitutes
the catalytically competent state. In the electronically excited state, light-induced
chromophore twisting may be enhanced, activating internal acid-base chemistry that
facilitates backbone cleavage to enlarge the chromophore. In this way, a softer active
site appears to be coupled to a mechanism involving concerted carbon acid deprotonation
and beta-elimination. Dynamics-driven hinge migration may represent a more general
platform for the evolution of novel enzyme activities by tuning motions in the active
site.
References:
1. Kim, H., Zou, T., Modi, C., Doerner, K., Grunkemeyer, T. J., Chen, L., Fromme,
R., Matz, M. V., Ozkan, B., Wachter, R. M. (2015). A Hinge Migration Mechanism Unlocks
the Evolution of Green-to-Red Photoconversion in GFP-like Proteins. Structure 23,
34-43.
2. Kim, H., Grunkemeyer, T. J., Modi, C., Chen, L., Fromme, R., Matz, M. V., Wachter,
R. M. (2013), Acid-Base Catalysis and Crystal Structures of a Least-Evolved Ancestral
GFP-like Protein Undergoing Green-to-Red Photoconversion. Biochemistry 52, 8048-8059.
PH-041
Quercetin effect on the stability and regeneration of the G-protein-coupled receptor
rhodopsin
Maria Guadalupe Herrera Hernández1,3, Xiaoyun Dong1, Cecylia S. Lupala2, Juan J. Perez2,
Pere Garriga1
1GBMI, Centre de Biotecnologia Molecular, Universitat Politecnica de Catalunya, 2GBMI,
ETSEIB, Universitat Politècnica de Catalunya, 3Unidad de Biotecnología. Campo Experimental
Bajío (INIFAP)
G-protein coupled receptors (GPCRs) are transmembrane heptahelical receptors that
constitute a large and widespread family of signal transduction proteins. A number
of extracellular ligands, ranging from small molecules to GPCR-binding proteins, have
been proposed as good candidates for drug design. The binding of an agonist to a GPCR
causes a conformational change in the receptor that leads to its activated functional
state. Rhodopsin, the membrane receptor responsible for photoreception in the vertebrate
retina, is a prototypical GPCR and has been extensively used in structural, biochemical
and biophysical studies of this class of receptors. Different small molecules have
been described to be capable of binding to rhodopsin. In addition, mutations in rhodopsin
have been associated with retinal diseases and efforts have been carried out in order
to find potential ligands that can offset the effect of these mutations. Cyanidins,
a group of flavonoids within the larger family of polyphenols, have been reported
to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration
intermediates. The aim of the current study was to evaluate the effect of the flavonoid
quercetin on the conformational properties of both native bovine rhodopsin and heterologously
expressed recombinant rhodopsin. Rhodopsin was purified from bovine retinas by immunoaffinity
chromatography, and photobleaching, thermal stability, metarhodopsin II decay and
chromophore regeneration assays were carried out in the absence or in the presence
of 1µM quercetin. For recombinant rhodopsin, a plasmid encoding wild-type opsin was
transfected into mammalian COS-1 cells, in the absence or in the presence of 1µM quercetin,
harvested, regenerated with 11-cis-retinal, or 9-cis-retinal, and subsequently purified
in dodecyl maltoside solution. No differences in photobleaching behavior, upon illumination,
could be detected in the purified quercetin-containing samples compared to those in
the absence of this flavonoid. In the case of rhodopsin, and the recombinant wild-type
protein regenerated with 11-cis-retinal, quercetin did not significantly alter the
thermal stability and rate of regeneration of the purified proteins under our experimental
conditions. However, a two-fold increase in the thermal stability and a 40% increase
in chromophore regeneration were observed for the recombinant wild-type protein regenerated
with 9-cis-retinal in the presence of quercetin. In contrast, the presence of quercetin
did not alter the electrophoretic and basic spectroscopic properties of rhodopsin,
or those of the recombinant wild-type protein, suggesting no important structural
alterations as a result of quercetin binding to the receptor. The positive effect
of quercetin on the stability, and chromophore regeneration of rhodopsin, could be
potentially used to counteract the effect of naturally-occurring misfolding mutations
in rhodopsin. Thus, quercetin could help stabilizing rhodopsin mutants associated
with retinal diseases such as retinitis pigmentosa. Furthermore, docking of the ligand,
carried out on the crystallographic structure of rhodopsin (entry 1GZM), reveals several
favorable sites for quercetin binding. One of this would be compatible with 9-cis-retinal
suggesting a complementary binding to the receptor of this isomer which would not
be compatible with 11-cis-retinal binding.
PH-042
Identification of prospective allosteric sites of p38 by computational methods
Patricia Gomez-Gutierrez1, Juan Jesus Perez1
1Departament d’Enginyeria Química (ETSEIB), Universitat Politècnica de Catalunya
Identification of prospective allosteric sites of p38 by computational methods Patricia
Gomez-Gutierrez and Juan J. Perez Grup de Biotecnologia Molecular i Industrial, Centre
de Biotecnologia Molecular Departament d’Enginyeria Química (ETSEIB), Universitat
Politècnica de Catalunya, Barcelona, Spain. Tel: 93 4016679 Protein function is intrinsically
associated with structural flexibility, so that understanding the functional properties
of proteins requires going beyond the static picture produced by X-ray diffraction
studies. Structural flexibility can also be interpreted as a dynamic exchange between
different conformational states with low energy barriers at room temperature. Allosterism
is a mechanism to regulate protein function associated with the plasticity exhibited
by proteins. Allosteric sites can be considered transient cavities that can be occupied
by a small molecule with the subsequent modulation of the protein plasticity. Occupation
of these sites may modify the affinity of the protein for its native substrate that
can be positive when the affinity increases or negative when the affinity decreases.
Allosterism can be used for the design of non-competitive ligands as new therapeutic
agents. This mechanism of activity modulation is particularly interesting for those
targets that use a common substrate for activation, like in the case of kinases to
search for selective compounds. Proteins can be viewed in solution as an ensemble
of diverse energy accessible conformations. Binding of an allosteric ligand produces
a redistribution of the population of the diverse conformational states, which at
the end modulate the affinity of the native substrate. Allosteric sites can be characterized
using computational methods by ensemble docking. It consist of characterize a set
of structures that represent the accessible conformations of a protein that can then
be used to perform virtual screening. In the present work we have studied prospective
allosteric sites of p38 using computational methods. The protein is a member of the
mitogen-activated protein kinases (MAPKs), a highly regulated group of enzymes that
control a variety of physiological processes, including mitosis, gene expression,
apoptosis and metabolism movement among others. The conformational profile of p38
was assessed using a 4 us trajectory of accelerated molecular dynamics as sampling
technique in explicit solvent. We used as starting structure the apo- form of p38
in its inactive conformation (entry 1P38). The conformational features of the protein
were assessed through the analysis of the variance of the most flexible regions of
the protein using principal component analysis. The snapshots of the trajectory were
projected onto the two principal components. Subsequent cluster analysis permitted
us to select a few structures for further studies. Specifically, prospective biding
sites were identified using a hydrophobic probe as implemented in the SiteMap program.
The results show previously described regulatory sites and some new prospective ones.
PH-043
Hydrogen/deuterium exchange-mass spectrometry provides clues on the mechanism of action
of Min E
Maria T. Villar1, Kyung-Tae Park2, Joe Lutkenhaus2, Antonio Artigues1
1Department of Biochemistry and Molecular Biology, 2Department of Microbiology, Molecular
Genetics & Immunology
Cell division in most bacteria is initiated by the formation of the Z ring, an essential
cytoskeletal element that serves as a scaffold for the cytokinesis machinery, at the
mid body of the cell. In E coli the spatial location of the Z ring is regulated by
the Min protein system, comprised by three major proteins: MinC, MinD and MinE. The
dynamic interaction between these proteins results in the formation of an oscillating
protein gradient between the poles of the cell. This oscillation determines the position
of the formation of the Z ring. Many aspects of this simple mechanism are beginning
to be understood. In particular, the conformational changes associated with the interaction
of the three Min proteins between them and with the cell membrane, are of especial
interest. Hydrogen/deuterium exchange mass spectrometry (HDX MS) is a sensitive technique
for the detection of changes in protein conformation and dynamics. The main advantages
of this methodology are the ability to study native proteins in solution, the requirement
for low protein concentrations, the potential to discriminate multiple coexisting
conformations, and the lack of an upper limit to the size of protein to be analyzed.
Here we use HDX MS to analyze the dynamics of the wild type MinE and of its inactive
double mutant D45A D49A. Our results show significant differences in the rates of
exchange and in the total amount of deuterium exchanged at the end of the reaction
between these two forms of MinE. The wild type protein exchanges most of the amide
hydrogen during the first few seconds of initiation of the exchange reaction. On the
other hand, the mutant protein exchanges only 50% of the total amide hydrogen atoms
during the first seconds of initiation of the exchange, and the remaining 50% amide
hydrogen atoms are exchanged more slowly during the next few minutes of the reaction.
Our data are consistent with the existence of a highly flexible structure for the
wild type protein and the coexistence of at least two rigid conformations for the
double mutant that are undergoing a cooperative transition. Interestingly, the central
β-sheet forming the interface between the two subunits is protected against exchange
on both proteins. These results provide insights into the conformational changes that
MinE undergoes during its interaction with MinD.
PH-044
Biased signalling and heteromization of the Dopamine D2 receptor in Schizophrenia
and Parkinson’s disease
Pablo Herrera Nieto1, James Dalton1_, Jesús Giraldo1_
1Universidad Autónoma de Barcelona
Biased signalling and heteromization of the Dopamine D2 receptor in Schizophrenia
and Parkinson’s disease As a significant component of dopamine signalling in the brain,
the dopamine D2 receptor (D2R), a member of the Class A GPCR family, is an important
target in the treatment of neurological conditions such as schizophrenia and Parkinson’s
disease. D2R shows a variety of signalling pathways through G proteins, including
adenylyl cyclase inhibition, Gβγpotentiation of adenylyl cyclase 2, and ERK kinase
activation, in addition to β-arrestin recruitment,. These pathways are differentially
activated by some agonists and it has been suggested that D2R ligands with Gαi/o antagonist
and β-arrestin agonist activity may have anti-psychotic behavioural activity with
reduced extra-pyramidal side effects. D2R has also been found to form homodimers or
higher-order hetero-oligomers with other GPCRs, which may modulate D2R conformation
and activity, thus constituting an additional form of allosteric receptor regulation.
Based on these findings, we have computationally modelled the full-length structure
of D2R, including its long intracellular loop 3 (ICL3) that is 130+ residues in length
and absent in all homologous GPCR crystal structures. Using state-of-the-art tools,
such as ROSETTA for ab initio protein folding and ACEMD for micro-second+ molecular
dynamics (MD) simulations we have successfully de novo folded ICL3, which primarily
consists of extensions to transmembrane helices (TMH) 5 and 6 and an intervening disordered
histidine/proline-rich region, which is highly flexible. The latter is observed to
interact with other receptor intracellular loops (ICL1 and ICL2) and appears to restrict
access to the G-protein binding-site. In addition, we have docked a structurally diverse
collection of 14 ligands (biased agonists, antagonists and allosteric modulators)
into our D2R model and observed characteristic binding patterns suggestive of different
biased signalling mechanisms. Finally, through protein-protein docking with ROSETTADOCK,
we have generated a complete heterodimer model of D2R with the Adenosine A2A receptor
(AA2AR), where a mutual interface is formed between their respective TMHs 4 and 5,
as well as an association between the C-terminus of AA2AR and ICL3 of D2R. This may
be a particularly relevant biological complex in the treatment of Parkinson’s disease
where antagonists of AA2AR have been shown to ameliorate disease effects, potentially
through direct interaction with D2R.
PH-045
Bis-ANS as a tool to monitor conformational changes upon assembly of binary and ternary
complexes of eIF4E, 4E-BP1 inhibitory protein, and the mRNA 5’cap
Anna Modrak-Wojcik1, Monika Wisniewska1, Ryszard Stolarski1
1Division of Biophysics, Faculty of Physics, University of Warsaw
Specific recognition of the mRNA 5’ terminal cap structure by the eukaryotic initiation
factor eIF4E is the first and rate-limiting step in the cap-dependent translation.
Small 4E-binding proteins, 4E-BP1, 4E-BP2, and 4E-BP3, inhibit the translation initiation
by competing with eIF4G initiation factor for the same binding site, and by blocking
the assembly of the translation machinery [1]. Our recent studies revealed intricate
cooperativity between the cap and 4E-BP1 binding sites of eIF4E [2]. Here, we applied
a fluorescent dye, 4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonate (bis-ANS) to investigate
conformational changes upon assembly of binary and ternary complexes composed of human
eIF4E, 4E-BP1, and the mRNA 5’cap analogue, m7GTP. The fluorescence quantum yield
of bis-ANS increases significantly upon binding to hydrophobic sites of proteins,
making the probe a convenient tool to determine the accessibility to hydrophobic surfaces,
and to monitor structural reorganisation of macromolecules [3]. We characterised the
interaction of bis-ANS with eIF4E and 4E-BP1 by fluorescence titration. The association
processes takes up to several hours until the saturation of the fluorescence signal
is achieved, reflecting high flexibility of the protein structures. The association
constants Kas of eIF4E/bis-ANS complexes are very high for the non-specific interaction.
The Kas values for eIF4E/bis-ANS and eIF4E/4E-BP1/bis-ANS are similar (∼107 M−1),
whereas the presence of m7GTP results in ca. 5-fold weaker binding of the probe to
eIF4E. The affinity of bis-ANS for 4E-BP1 is ∼10-fold lower than that for eIF4E. We
found no effect of either m7GTP or 4E-BP1 on the fluorescence of bis-ANS in complex
with eIF4E, thus indicating lack of conformational changes around the probe on eIF4E/m7GTP
or eIF4E/4E-BP1 complex formation. It also testifies that bis-ANS does not bind to
the cap-binding site, despite the hydrophobic nature of this eIF4E region. On the
contrary, addition of m7GTP to the eIF4E/4E-BP1/bis-ANS complex causes an increase
of the probe fluorescence, which indicates differences in the structural reorganisation
in the binary, m7GTP/eIF4E, compared with the ternary, m7GTP/eIF4E/4E-BP1, complexes,
and confirms the spatial cooperation between the cap and 4E-BP1 binding sites. We
also observed an increase of fluorescence for bis-ANS bound to 4E-BP1 in the presence
of eIF4E, pointing out that 4E-BP1 partially folds upon association with eIF4E. In
summary, our results provide a deeper insight into the structural aspects of the molecular
interaction at early stages of the cap-dependent translation.
Acknowledgements:
This work was supported by the BST 170000/BF project from University of Warsaw
[1] N. Sonenberg, A.G. Hinnebusch, Cell 136 (2009) 731-745
[2] A. Modrak-Wojcik, M. Gorka, K. Niedzwiecka, K. Zdanowski, J. Zuberek, A. Niedzwiecka
and R. Stolarski., FEBS Letters 587 (2013) 3928–3934
[3] A. Hawe, M. Sutter, and W Jiskoot, Pharmaceutical Research 25 (2008) 1487-1499
PH-046
Mapping of Thrombin - Beta2Glycoprotein I Interaction Sites
Laura Acquasaliente1, Simone Tescari1, Daniele Peterle1, Giulia Pontarollo1, Vittorio
Pengo2, Vincenzo De Filippis1
1Department of Pharmaceutical and Pharmacological Sciences, University of Padua, 2Department
of Cardiac, Thoracic and Vascular Sciences, University of Padua
Background: Beta2-Glycoprotein (B2GpI) is a protein abundantly present in human plasma
and highly conserved in all mammals. B2GpI has been identified as the major antigen
in the antiphospholipid syndrome (APS), a severe thrombotic autoimmune disease. Despite
its importance in the pathogenesis of APS, the physiological role of B2GpI is still
elusive. In a previous work we have demonstrated that B2GpI significantly prolongs
the clotting time in fibrin generation assays, and inhibits aggregation of gel-filtered
platelets (IC50=0.36uM), either isolated or in whole blood, by inhibiting cleavage
of PAR1 on intact platelets (IC50=0.32uM) and in solution. Importantly, B2GpI does
not alter the ability of thrombin (FIIa) to generate the anticoagulant protein C,
with or without thrombomodulin added. Hence, we concluded that B2GpI inhibits the
key procoagulant properties of FIIa, without affecting its unique anticoagulant function.
We also proposed that B2GpI, together with other more efficient anticoagulant pathways
such as thrombomodulin- FIIa -protein C and antithrombin III- FIIa, may function as
a mild anticoagulant in vivo especially in those compartments were the efficacy of
thrombomodulin is limited, as in the large vessels, or is even absent, as in the brain
vasculature. Aims: Lacking the threedimensional structure of B2GpI-thrombin complex,
the aim of this work is to identify the peptide regions either on thrombin and B2GpI
involved in complex formation. Results: Data obtained by fluorescence and surface
plasmon resonance (SPR) indicated that B2GpI interacts whit FIIa whit physiological
affinity (Kd=43 ± 4nM). Kd values calculated by reverting the interacting systems
are very similar to each other (Kd=98 ± 9nM), suggesting that B2GpI in the mobile
phase has a conformation which is competent for the binding to immobilized FIIa. The
affinity of FIIa for immobilized B2GpI is markedly decreased by increased ionic strength
(i.e. Kd increases by 50-fold going from 0.1 M to 0.4 M), suggesting the electrostatic
interactions play a key role in FIIa - B2GpI recognition. Filling/inactivation or
perturbation of FIIa active site does not alter the affinity of FIIa for immobilized
B2GpI, confirming that the active site is not involved in the interaction. Mapping
of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, GpIbalpha,
HD1 aptamer) and thrombin analogues having the exosites variably compromised (i.e.
prothrombin, prethrombin-2, alpha-thrombin), reveals that the positively charged exosite-II
of FIIa plays a key role in B2GpI binding. From the docking model of the bB2GpI-thrombin
complex, we identified a highly negatively charged segment 219-232 in domain V of
B2GpI interacting with positively charged pathes in thrombin exosite II. The synthetic
peptide B2GpI(219-232) was able to bind to FIIa with an affinity (Kd=38 ± 9nM) comparable
to that of full-length B2GpI, deduced from fluorescence or SPR measurements and to
compete in SPR measueremnts with the binding of full-length B2GpI to thrombin. Hence,
combining experimental and theoretical data, we obtained a reliable model of the B2GpI-thrombin
complex.
PH-047
Dynamical Variability in the Clan MA of Metalloproteases
Henrique F. Carvalho1,2, Ana Cecília A. Roque1, Olga Iranzo3, Ricardo J. F. Branco1
1UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova Lisboa, 2ITQB
António Xavier, Universidade Nova de Lisboa, 3Aix Marseille Université, Centrale Marseille,
CNRS, iSm2 UMR 7313
Metalloproteases are one of the most diverse types of proteases, presenting a wide
range of folds and catalytic metal ions. In the case of the MEROPS MA clan, where
most of the known metalloproteases are grouped based on the consensus HEXXH sequence
motif, a single catalytic zinc ion and common fold architecture [1]. Despite these
common features, members from distinct families present distinct domain composition
and topology. Given our interest in developing new tailor-made metalloproteases for
bioengineering applications, an in-depth understanding of the factors governing their
function is required. Protein internal dynamics includes the space of functionally-relevant
structural changes occurring during an enzymatic reaction, and there is an increasing
understanding on how it relates with protein sequence and structure evolution. Therefore,
we have recently assessed how the structural heterogeneity of metalloproteases relates
with the similarity of their dynamical profiles [2]. First, the dynamical profile
of the clan MA type protein thermolysin, derived from the Anisotropic Network Model,
was evaluated and compared with those obtained from principal component (PC) analysis
of a set of 112 crystallographic structures and essential dynamics (ED) analysis of
a 20 ns molecular dynamics simulation trajectory [3]. A close correspondence was obtained
between normal modes (NM) derived from the coarse-grained model and experimentally-observed
conformational changes (RMSIP between NM1-NM3 and PC1 of 0.81), corresponding to functionally-relevant
hinge bending motions that were shown to be encoded in the internal dynamics of the
protein (cumulative overlap of ED1-ED3 and PC1 of 0.85). Next, dynamics-based comparison
methods that employ a related coarse-grained model (β-Gaussian Elastic Network Model)
was made for a representative set of 13 MA clan members [4], allowing for a quantitative
description of its structural and dynamical variability. Although members are structurally
similar (87% pairs with DaliLite Z-score > 2.0), they nonetheless present distinct
dynamical profiles (69% of pairs with ALADYN P-value > 0.02), with no identified correlation
between structural and dynamical similarity. For cases where high dynamical similarity
was observed, the respective modes corresponded to hinge-bending motions encompassing
regions close to the active site. Further inspection of the produced alignments indicates
that for MA clan metalloproteases, conservation of internal dynamics has a functional
basis, namely the need for maintaining proper intermolecular interactions between
the protein and respective substrate. Previously unnoticed dynamical similarity between
clan members Botulinum Neurotoxin Type A, Leishmanolysin and Carboxypeptidase Pfu
was also found. Together, these results suggest that distinct selective pressure mechanisms
acted on metalloprotease structure and dynamics through the course of evolution. This
work shows how new insights on metalloprotease function and evolution can be assessed
with comparison schemes that incorporate additional information of protein dynamics.
[1] Rawlings ND, Waller M, Barrett AJ, Bateman A. Nucleic Acids Res. 2014; 42: D503–9.
[2] Carvalho HF., Roque ACA., Iranzo O., Branco, RJF, submitted.
[3] Bakan A, Meireles LM, Bahar I. Bioinformatics. 2011; 27: 1575–7.
[4] Potestio R, Aleksiev T, Pontiggia F, Cozzini S, Micheletti C. Nucleic Acids Res.
2010;38: W41–5.
PH-048
X-ray crystallographic analysis of cold-adapted and thermostable glucokinase
Tokuro Oda1, Naoki Fuchita1, Hiroyuki Motoshima1, Keiichi Watanabe1,
1Department of Applied Biochemistry and Food Science, Saga University
Glucokinase from Antarctic psychrotroph Pseudoalteromonas sp. AS-131 (PsGK) has a
higher specific activity at low temperatures and a higher thermal stability than its
mesophilic counterpart from E. coli (EcGK). In order to elucidate the structural basis
for cold–adaptation and thermal stabilization of PsGK, we have determined the crystal
structure of PsGK at 1.69 Å and compared it with the EcGK structure. PsGK is a homodimer
of the subunit of 328 amino acid residues. Each subunit consists of two domains, a
small α/β domain (residues 7–125 and 314–328) and a large α + β domain (residues 126–313).
The active site is located in a cleft formed between the two domains. The identity
of amino acid sequence between PsGK and EcGK was 36%, but three dimensional structures
of them are very similar to each other, having the conserved catalytic residues and
substrate–binding residues. The analysis of the main–chain temperature factors revealed
that the regions of small domain and the hinge region connecting two domains of PsGK
showed higher temperature factors with a lower number of intramolecular hydrogen bonds
and ionic interactions than the corresponding regions of EcGK. However, the large
domain regions of PsGK showed lower temperature factors with a higher number of intramolecular
hydrogen bonds than EcGK. Furthermore, the atomic temperature factors of catalytic
Asp112 on the small domain were higher, but those of glucose-binding Glu169, His172,
and Glu199 on the large domain were lower than EcGK. These results suggest that highly
flexible hinge region and the catalytic residue on the small domain of PsGK may contribute
to its cold-adaptation, namely higher activity at low temperatures, whereas a more
rigid structure of the large domain of PsGK stabilizes its overall structure more
strongly than EcGK.
PI-001
Genetic engineering of new formate dehydrogenases for cofactor regeneration
Anastasia Alekseeva1,2, Irina Dolina2,3, Ivan Kargov2,3, Svyatoslav Savin2,3, Vladimir
Tishkov1,2,3
1A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, 2Innovations and
High Technologies MSU Ltd, 3Chemistry Faculty, M.V. Lomonosov Moscow State University
Nowadays non-waste technologies in synthetic chemistry become more and more popular.
Such processes are often carried out using different enzymes. Dehydrogenases represent
the large group of enzymes, which are widely used in synthesis of chiral compounds
and other useful molecules. Such enzymes need NADH or NADPH as a cofactor and due
to high cost of reduced coenzymes a cofactor regeneration system is an obligate part
in such kind of processes. It was shown that formate dehydrogenase (FDH, EC 1.2.1.2.)
is one of the best enzymes for NAD(P)H regeneration. FDH catalyses the reaction of
formate oxidation to carbon dioxide coupled with reduction of NAD(P)+ to NAD(P)H.
The main advantages of FDH are the irreversibility of catalyzed reaction, low price
of formate ion and wide pH optimum of activity. Our laboratory has the largest collection
of formate dehydrogenases from different sources. Many FDH genes from bacteria, yeasts
and plants were cloned and enzymes were expressed in active and soluble forms. Mutant
formate dehydrogenases from bacterium Pseudomonas sp.101 show the highest thermal
stability as well as activity in comparison with other reported formate dehydrogenases.
Now we have focused on eukaryotic genes. The recombinant enzymes from soya Glycine
max (SoyFDH), Arabidopsis thaliana (AthFDH), moss Physcomitrella patens (PpaFDH) and
yeast Ogataea parapolymorpha (OpaFDH) were obtained by genetic engineering methods.
It was revealed, that SoyFDH has the best Michaelis constants among all known FDHs,
but it’s less thermally stable compared to other FDHs. New mutant forms of SoyFDH
with excellent catalytic characteristics and high thermal stability were obtained
by protein engineering. Other enzymes (AthFDH, PpaFDH and OpaFDH) are comparable in
their stability with majority of bacterial enzymes (but not with PseFDH), so all the
new obtained FDHs can be successfully used for cofactor regeneration.
This work was supported by grant of Russian President MK-2304.2014.4 and Russian Foundation
for Basic Research (grant RFBR 14-04-01665 A and14-04-01625 A).
PI-002
Mutations of a conserved tryptophan residue of the TEM-1 ß-lactamase
F Ece Altinisik1, F Gizem Avci1, Berna Sariyar Akbulut1, Elif Özkirimli Ölmez3, Didem
Vardar Ulu2, Ipek Karacan1, Duygu Sentürk1
1Marmara University, 2Wellesley College, 3Bogaziçi University
Antibiotics are essential therapeutic drugs widely used in the treatment of bacterial
infections. Unfortunately, misuse of these drugs resulted in the development of bacterial
defense mechanisms. Β-lactamase synthesis is among these mechanisms that renders β-lactam
antibiotics ineffective. Understanding the dynamic behavior of this enzyme is an important
step in controlling its activity. In a former study, the importance of highly conserved
W229 in modulating the hinge type H10 motion was reported. In the light of this information,
mutant TEM-1 β-lactamase enzymes with W229A, W229F and W229Y substitutions were constructed.
Wild-type and mutant TEM-1 β-lactamases purified with Ni2+-affinity chromatography
were subjected to enzyme assay using CENTA as the substrate. With W229F and W229Y
mutations, the remaining activity was approximately 10% of the initial activity. However
with the W229A mutation, activity was totally lost. Structural studies of the W229A
mutant with CD and florescence spectroscopy indicated that there was no major change
in the overall structure. However this mutation disrupted the interactions of W229
which resulted in an increase in the flexibility of this region of the protein.
This project was supported by TÜBİTAK project no 113M533.
PI-003
Light-switchable Zn2+ binding proteins to study the role of intracellular Zn2+ signaling
Stijn Aper1, Maarten Merkx1
1Eindhoven University of Technology
Zn2+ plays an important catalytic and structural role in many fundamental cellular
processes and its homeostasis is tightly controlled. Recently, free Zn2+ has also
been suggested to act as an intracellular signaling molecule. To get increased understanding
of the signaling role of Zn2+ we are developing light-switchable Zn2+ binding proteins
to perturb the intracellular Zn2+ concentration using light. These protein switches
consist of two light-responsive Vivid domains and the Zn2+ binding domains Atox1 and
WD4, linked together with flexible peptide linkers. In the dark, Zn2+ is tightly bound
in between the two Zn2+ binding proteins. Light-induced dimerization of the Vivid
proteins disrupts this interaction and thus results in Zn2+ release. The fluorescent
proteins Cerulean and Citrine were attached to the Vivid domains to allow the different
conformational states of the protein switch to be monitored using FRET. Zn2+ titrations
revealed a 3-fold decrease in Zn2+ affinity going from dark- to light-state for the
initial design, which was further improved to 10-fold by optimizing the linkers between
the protein domains. In addition, the Zn2+ affinities of both states were tuned to
be optimal for intracellular applications. Switching between the high affinity dark-state
and the low affinity light-state was found to be reversible for at least two light-dark
cycles. Following the in vitro characterization, we are currently assessing the performance
of this genetically encoded ‘caged’ Zn2+ in mammalian cells.
PI-004
Proteins as supramolecular building blocks: engineering nanoscale structures
Helen Ashmead1,2,3, Leonardo Negron1, Jack Sissons6, Kyle Webster6, Vic Arcus2,4,
Juliet Gerrard1,2,5
1Callaghan Innovation, 2Biomolecular Interaction Centre, University of Canterbury,
3School of Biological Sciences, University of Canterbury, 4Faculty of Science & Engineering,
University of Waikato, 5School of Biological Sciences, University of Auckland, 6School
of Biological Sciences, Victoria University
Proteins hold great promise in forming complex nanoscale structures which could be
used in the development of new nanomaterials, devices, biosensors, electronics and
pharmaceuticals. The potential to produce nanomaterials from proteins is well supported
by the numerous examples of self-assembling proteins found in nature. We are exploring
self-assembling proteins for use as supramolecular building blocks, or tectons, specifically
the N-terminal domain of a DNA binding protein (Nterm-Lsr2) and a typical 2-cys peroxiredoxin
(hsPrx3). Non-native forms of these proteins have been designed undergo self-assembly
into supramolecular structures in a controllable manner. Self-assembly of Nterm-Lsr2
is initiated via proteolytic cleavage, thereby allowing us to generate supramolecular
assemblies in response to a specific trigger. We will show that the degree of oligomerisation
can be controlled by variations in environmental conditions such as pH and protein
concentration. Furthermore, via protein engineering, we have introduced a new “switch”
for oligomerisation via enteropeptidase cleavage. The new construct of Nterm-Lsr2
can be activated and assembled in a controlled fashion and provides some ability to
alter the ratio of higher ordered structures formed. hsPrx3 has been shown to oligomerise
into dimers, toroids, stacks and tubes in response to specific triggers such as pH
and redox state. In this work we have utilised the histidine tag to further control
the assembly of this versatile protein tecton. We will show that minute variations
in pH can induce oligomersation of hsPrx3 toroids into stacks and tubes. Furthermore,
by utilising the histidine tag as a ligand we can bind divalent metals to these supramolecular
structures. This not only drives the formation of higher ordered oligomers but also
provides a facile route which may facilitate the functionalisation of these protein
nanoscale structures after they have been assembled.
PI-005
A Structure Based Approach to Engineering Contraceptive Vaccine Antigens
Danielle Basore1,2, Rajesh Naz5, Scott Michael6, Sharon Isern6, Benjamin Wright3,
Katie Saporita1, Donna Crone1, Christopher Bystroff1,2,4
1Biological Sciences, Rensselaer Polytechnic Institute, 2CBIS, Rensselaer Polytechnic
Institute, 3Chemical and Biological Engineering, Rensselaer Polytechnic Institute,
4Computer Science, Rensselaer Polytechnic Institute, 5Obstetrics and Gynecology, West
Virginia University, 6Biological Science, Florida Gulf Coast University
Unintended pregnancy is a worldwide public health concern, with 85 million pregnancies
being classed as unintended in 2012. The magnitude of this number clearly indicates
an unmet need in terms of contraception. Methods that are currently available are
effective, but exhibit many problems. Side effects, ease of use, cost, and availability
are all concerns. We propose a contraceptive vaccine that would be safe, effective,
long-lasting, cheap, and reversible. Our vaccine would prevent pregnancy by targeting
sperm with antibodies raised in the woman’s body. Several approaches have been taken
to developing a contraceptive vaccine in recent years. The most successful so far
has been using human chorionic gonadotropin (hCG), a hormone produced during pregnancy,
as an antigen. The hCG vaccine progressed to phase 2 clinical trials, but only displayed
an 80% efficacy, which is insufficient for a contraceptive. Our lab uses a structure
based approach to the design of an anti-sperm antigenic protein. We believe this will
raise a more vigorous immune response that will produce a longer lasting titer. The
CatSper complex is a heterotetrameric calcium channel found in the tail region of
sperm. Each subunit of the complex contains an exposed loop known as the P-loop. The
P-loop is unique on the surface of sperm because it is not glycosylated, allowing
antibodies to potentially recognize and bind it. YLP12 is a twelve residue peptide
that mimics the glycans in the glycocalyx of sperm. YLP12 is a member of the FliTRX
library, and in mice, produced protective titers that were reversible both voluntarily
and involuntarily. Our designs will introduce these two potential antigens into a
loop of the L1 protein of Human Papilloma Virus. L1 spontaneously assembles into virus
like particles, and will aid in the production of a robust immune response.
PI-006
Protein carriers for passage of the Blood–Brain Barrier
Sinisa Bjelic1
1Department of Chemistry and Biomedical Sciences, Linnaeus University
Medical solutions that help protein therapeutics accumulate into the brain are crucial
for future treatment of neurological disorders. Biodrugs have a tremendous potential
to treat disorders of the nervous system, but their efficiency has been severely restricted.
To reach the brain all drugs must traverse the blood–brain barrier (BBB) — a permeable
wall that separates blood from the brain — whose main function is to protect the nervous
system from environmental influences of bacteria and toxins. Unfortunately the BBB
is also the culprit that effectively blocks access to therapeutics required for treatment
of neurological diseases. A way to boost exposure of therapeutics across the BBB is
to piggyback onto the transferrin receptor, a multidomain protein anchored in the
membrane, which is involved in the physiological facilitation of iron uptake. Here
I present research that aims at successfully developing potent protein carriers for
transferrin receptor-mediated passage of the BBB by using computational protein design
in combination with yeast display methodology for hit validation and optimization.
The long-term goal is to couple therapeutics — as for example drugs against Alzheimer’s
— to the designed carriers to increase the brain uptake and cure neurological disorders.
PI-007
Medium-throughput multistep purification of coagulation factor VIIa
Jais R. Bjelke1, Gorm Andersen1, Henrik Østergaard1, Laust B. Johnsen1, Anette A.
Pedersen1, Tina H. Glue1
1Global Research Unit, Novo Nordisk
There is a need of medium-to-high throughput purification of low-titre recombinant
protein variants for screening to identify the final biopharmaceutical lead. Such
proteins include coagulation factors to be used for treatment of haemophilia and other
bleeding disorders. At Novo Nordisk we have established a platform for production
of recombinant coagulation factor VIIa variants, which include a spectrum of single-point
mutations to large domain insertions. The variants were produced using transiently
transfected HEK293F, HKB11 or CHOEBNALT85 (QMCF Technology) suspension cells. Harvest
cultivations were typical in the range of 0.3- to 1L. A 3-step continuous, multistep
purification method was implemented on ÄKTAxpress systems (GE Healthcare). The interlinked
process steps include capture using an immunoaffinity column, polish, concentration
and buffer exchange using an anion-exchange column and proteolytic activation of the
zymogen variant forms using a coagulation factor Xa-immobilized column. Buffers were
designed such that elution from the capture column was aligned with binding conditions
on the polish column to avoid a desalting step in-between. The following and final
enzymatic activation was optimized with regards to flow rate to ensure full conversion
while minimizing unwanted secondary cleavages in factor VIIa. The final products were
fractionated in sharp chromatographic peaks ready for characterization. HPLC and SDS-PAGE
analyses showed a solid quality of the produced variants and more than 800 variants
have been produced in sub mg scale using the outlined method.
PI-008
Biomimetic Sequestration of CO2: reprogramming the B1 domain of protein g through
a combined computational and experimental approach
Esra Bozkurt1, Ruud Hovius1, Thereza A. Soares2, Ursula Rothlisberger1
1École Polytechnique Fédérale de Lausanne, 2Federal University of Pernambuco
Protein engineering is a powerful tool to generate highly specific enzymes for biomimetic
production of chemicals. Among many applications, the development of enzymes to accelerate
carbon dioxide fixation is a possible route to limit CO2 emission. In this project,
we are inspired by the ancient enzyme carbonic anhydrase which efficiently catalyzes
the reversible hydration of carbon dioxide in the presence of a zinc ion active site.1
To create an efficient biocatalyst, the engineered GB1 domain2 containing a His3Cys
Zn (II) binding site was used as a starting point.3 In subsequent work, B1 domains
comprising of His3Wat Zn (II) binding sites have been rationally designed to produce
carbonic anhydrase mimics. The re-engineering was accomplished through a series of
mutations to orient the zinc bound reactive species to form a hydrogen bond network
in the active site while retaining the native secondary structure. We performed classical
molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) simulations
and metadynamics, with the aim to explore potential catalytic roles of the re-engineered
B1 domains and to elaborate the reaction mechanism. Briefly, we introduced novel Zn
(II) binding sites into thermostable B1 domain. In parallel, experiments are underway.
Wild-type protein was expressed and purified. Structural and mutagenesis studies are
ongoing. The results emphasize the power of theoretical work to enable the mimicking
of Nature’s enzymes for desired catalytic functions.
PI-009
The roles of entropy and packing efficiency in determining protein-peptide interaction
affinities
Diego Caballero1,2, Corey O’Hern1,2,3,4, Lynne Regan2,5,6
1Physics, Yale University, 2Integrated Graduate Program in Physical and Engineering
Biology, Yale University, 3Mechanical Engineering and Materials Science, Yale University,
4Applied Physics, Yale University, 5Molecular Biophysics and Biochemistry, Yale University,
6Chemistry, Yale University
Despite many recent improvements in computational methods for protein design, we still
lack a quantitative and predictive understanding of the driving forces that control
protein stability, for example, we do not know the relative magnitudes of the side-chain
entropy, van der Waals contact interactions, and other enthalpic contributions to
the free energy of folded proteins. In addition, we cannot reliably predict the effects
of point mutations on enzyme specificity or sequence tolerance in ligand binding sites.
The tetratricopeptide repeat (TPR) motif is a common and versatile protein system
that has been used as a model to study protein-protein interactions. For example,
recent studies have experimentally measured the binding affinity and specificity for
different TPR binding pockets and peptide ligands and generated a ranking of the protein-peptide
pairs with the highest affinity. To gain a fundamental understanding of the interplay
between atomic close packing and fluctuations of side-chain conformations in protein-peptide
binding pairs, we performed all-atom Langevin Dynamics simulations of key residues
near the binding interface of TPR proteins and their cognate peptides. The Langevin
Dynamics simulations enabled us to calculate the entropy and potential energy of side
chain conformations in the presence of backbone fluctuations for each protein-peptide
pair. We compile rankings of the stability and affinity of mutant TPR-peptide structures
to those obtained from experimental studies. This research has enhanced our ability
to rationally manipulate protein-peptide interfaces. Advances from this research will
enable the design of TPR modules that specifically recognize biologically important
proteins.
PI-010
Monitoring protein-protein interactions using tripartite split-GFP complementation
assays
Stéphanie Cabantous1, Hau B. NGuyen3, Jean-Denis Pedelacq2, Faten Koraichi1, Anu Chaudhary3,
Kumkum Ganguly3, Meghan A. Lockard3, Gilles Favre1, Thomas C. Terwilliger3, Geoffrey
S. Waldo3
1Cancer Research Center of Toulouse, 2CNRS- IPBS, UMR 5089, 205 Route De Narbonne,
3Los Alamos National Laboratory, Los Alamos NM
Protein-fragment complementation assay (or PCA) is a powerful strategy for visualizing
protein-protein interactions in living cells. Previously described split-GFP based
sensors suffer from the poor solubility of individual PCA fragments in addition to
background signal originating from their spontaneous self-assembly (1). We developed
a new encoded genetic reporter called “tripartite split-GFP” for visualizing protein-protein
interactions in vitro and in living cells. The assay is based on tripartite association
between two twenty amino-acids long split-GFP tags, GFP10 and GFP11, fused to interacting
protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10
and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP (2). Using coiled-coils
and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia
coli. We extended our studies to mammalian cells and examine the FK-506 inhibition
of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and
their minimal effect on fusion protein behavior and solubility should enable new experiments
for monitoring protein-protein association by fluorescence and for screening modulators
of complex formation in cell-based assays.
References:
[1] Cabantous S., T. C. Terwilliger, et al. (2005). Protein tagging and detection
with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol
23(1): 102-107.
[2] Cabantous S, Nguyen HB, Pedelacq JD, Koraichi F, Chaudhary A, Ganguly K, Lockard
MA, Favre G, Terwilliger TC, Waldo GS. A new protein-protein interaction sensor based
on tripartite split-GFP association. Sci Rep 2013;3:2854.
PI-011
Role of residues Cys301 and Cys303 in the active site of human ALDH2.
Luis Francisco Calleja Castañeda1, José Salud Rodríguez Zavala1
1Instituto Nacional de Cardiología ’Ignacio Chávez’
Aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes to their corresponding
acids using NAD(P)+ as coenzyme. These enzymes are responsible for the detoxification
of lipid peroxidation products, which have been involved in the etiology and pathogenesis
of different diseases involving increments in oxidative stress. Recent data from our
group, showed that ALDH3A1 is resistant to inactivation by lipid peroxidation products,
even at concentrations 50-100 times higher than those required to inactivate ALDH1A1
and ALDH2. The amino acids sequence of the aldehyde-binding site of the three enzymes
was analyzed, and it was found that the enzymes susceptible to the effect of lipid
peroxidation products (ALDH1A1 and ALDH2), have Cys residues flanking the reactive
Cys (position 302), based on this criteria and considering that these aldehydes react
preferentially with cysteine, a mutant of ALDH2 was generated changing the Cys residues
adjacent to Cys302. The mutant ALDH2-Cys301Thr-Cys303Val, was resistant to the inactivation
by acrolein and 4-HNE, even at concentrations 1000-fold higher than those required
to inactivate ALDH2. However, the mutant presented values of Km 2, 5 and 50-fold higher
for acrolein, propionaldehyde and acetaldehyde, respectively, compared to the wild
type enzyme, but showed a catalytic efficiency similar to the parent enzyme. These
data revealed that Cys residues near to the reactive Cys in ALDH2 are important in
the inactivation process induced by lipid aldehydes, but also participate in determining
the specificity for the substrates in this enzyme.
PI-012
Small molecule-assisted shutoff: A widely applicable method for tunable and reversible
control of protein production
H. Kay Chung1, Conor Jacobs1, Yunwen Huo2, Jin Yang3, Stefanie Krumm4, Richard Plemper4,5,
Roger Tsien0, Michael Lin3
1Department of Biology, Stanford University, 2Department of Pediatrics, Stanford University,
3Department of Pharmacology, University of California San Diego, 4Department of Pediatrics,
Emory University, 5Institute for Biomedical Sciences, Georgia State University, 6Department
of Chemistry and Biochemistry, University of California San Diego, 7Howard Hughes
Medical Institute, University of California San Diego, 8Department of Bioengineering,
Stanford University
The ability to quickly control the production of specific proteins would be useful
in biomedical research and biotechnology. We describe Small Molecule-Assisted Shutoff
(SMASh), a technique in which proteins are fused to a self-excising degron and thereby
expressed in a minimally modified form by default. Degron removal is performed by
a cis-encoded hepatitis C virus (HCV) protease, so that applying clinically available
HCV protease inhibitors causes degron retention on subsequently synthesized protein
copies and suppresses further protein production. We find that SMASh allows reversible
and dose-dependent shutoff of various proteins with high dynamic range in multiple
cell types, including yeast. We also successfully use SMASh to confer drug responsiveness
onto a RNA virus for which no licensed drug inhibitors exist. As SMASh does not require
permanent fusion of a large domain, it should be useful when control over protein
production with minimal structural modification is desired. Furthermore, as SMASh
only uses a single tag and does not rely on modulating protein-protein interactions,
it should be easy to generalize to multiple biological contexts.
Figure 1.
Small Molecule-Assisted Shutoff (SMASh) concept and development. (a) SMASh concept.
Top, a protein of interest is fused to the SMASh tag via a HCV NS3 protease recognition
site. After protein folding, the SMASh tag is removed by its internal NS3 protease
activity, and is degraded due to an internal degron activity. Bottom, addition of
protease inhibitor induces the rapid degradation of subsequently synthesized copies
of the tagged protein, effectively shutting off further protein production.
Figure 2.
Proteins can be regulated by SMASh tags at either end dose-dependent manner (a) SMASh
can regulate YFP when fused to either terminus. SMASh-YFP or YFP-SMASh were expressed
in HEK293 cells in the absence or presence of ASV for 24 hours. Immunoblotting revealed
shutoff of YFP expression by asunaprevir (ASV) for both constructs. DMSO was used
as vehicle control. (β-actin served as a loading control. (b) Fluorescence microscopy
confirmed shutoff of YFP expression by ASV for both constructs. Scale bar, 50 pm.
(c) To test dose-dependent regulation of protein expression by SMASh, HEK293 cells
transfected with YFP-SMASh were cultured for 24 hours without or with ASV (15 pM to
15 µM) and YFP was detected by immunoblot. GAPDH served as loading control. Quantification
of YFP levels by immunoblot. Background-subtracted YFP signal was normalized to background-subtracted
GAPDH signal, and then plotted as a percent of the signal in the untreated condition
(n = 3, error bars represent standard deviations).
Figure 3.
SMASh functions in budding yeast. (a) SMASh-YFP or YFP-SMASh were expressed from the
strong constitutive GPD promoter in wild-type or drug efflux pump-deficient yeast
cells. The yeast cells were cultured in SD media in the absence or presence of ASV
for 24 hours. Immunoblotting revealed shutoff of YFP expression by ASV for both constructs.
DMSO was used as vehicle control. GAPDH served as a loading control. (b) Fluorescence
images of yeast cultures in (a) show that episomally-expressed YFP signal is controlled
in a drug-dependent manner. Imaging was done in SD media. Scale bar, 10 µm. (c) The
SMASh tag were inserted at the C-terminus of the endogenous SEC14 coding sequence,
and serial dilutions of cells were plated and incubated for 48 h at 30°C and 23°C
in the absence or presence of ASV (3 µM).
PI-013
Proof of principle for epitope-focused vaccine design
Bruno Correia1, John Bates2, Rebecca Loomis3, Chris Carrico4, Joseph Jardine5, David
Baker6, Roland Strong7, James Crowe3, Phillip Johnson4, William Schief1,6,7
1Institute of Bioengineering, Swiss Federal Institute of Technology Lausanne, 2Department
of Biochemistry, University of Washington, 3The Vanderbilt Vaccine Center, Vanderbilt
University Medical Center, 4The Children’s Hospital of Philadelphia Research Institute,
5Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 6Department of
Immunology and Microbial Science, The Scripps Research Institute, 7IAVI Neutralizing
Antibody Center, The Scripps Research Institute
Novel strategies for the development of efficacious vaccines are currently needed
for several serious global health threats (e.g. HIV, Flu, Ebola, Dengue, etc). A new
class of protein based immunogens for vaccine development has emerged, epitope-focused
immunogens, but in the past these have failed to deliver the expected outcome. Here,
we employed a new computational design methodology (Rosetta Fold From Loops or FFL)
to design epitope-focused immunogens. FFL was devised to insert structurally defined
functional sites into protein scaffolds. Throughout the FFL stages the structure of
the scaffold is folded and its sequence designed to stabilize the desired functional
conformation of the inserted site. We used FFL to design epitope-focused immunogens
for the Respiratory Syncytial Virus (RSV), for which despite the intense research
we are still lacking an approved vaccine. We designed three-helix bundles harboring
an RSV epitope, that was previously co-crystallized with the neutralizing antibody
motavizumab. The designs were thermodynamically stable (Tm > 100˚C) and showed extremely
high affinities to motavizumab (KD ≈ 30 pM). Structural characterization through x-ray
crystallography of antibody-bound and unbound scaffolds showed good agreement to the
computational models in the overall structure (rmsd - 1.2 Å) and exquisite mimicry
of the epitope region (rmsd - 0.4 Å), when compared to the peptide-epitope in complex
with motavizumab. The designed immunogens were used to immunize non-human primates
(NHP), and approximately 75% of the cohort developed RSV neutralizing activity, in
some instances with high potency. To evaluate the therapeutic relevance of the elicited
neutralization activity, we compared the NHP neutralization titers to those of human
sera after natural RSV infection, which generally yields protective levels of antibodies.
The neutralization potency of the best NHP responders was comparable to that of the
human sera. To better understand the features of the antibodies elicited, we isolated
several rhesus monoclonal antibodies (RhmAbs) from the animal that exhibited the most
potent neutralization. Two of the RhmAbs bound to the immunogen with very high affinity
(KD ≈ 3 pM) and were potent RSV neutralizers. Interestingly, these RhmAbs were approximately
10 fold more potent than the FDA-approved prophylactic antibody Palivizumab. Our results
provide the first proof-of-principle for epitope-focused vaccine design, and demonstrate
the power of the FFL computational methodology. We anticipate that FFL will be useful
for a variety of other challenges in the computational design of functional proteins.
Figure 1.
Schematic of nucleotide binding, exchange and hydrolysis in tubulin, and its coupling
to MT assembly. Exchange of GDP (orange) for GTP (magenta) at the E-site in b-tubulin
(blue) happens in the unpolymerized dimer (left). The active, GTPbound tubulin dimer
adds to a growing MT (right). Interaction of the incoming a-tubulin (green) with the
E-site nucleotide at the plus end of a MT (with b-tubulin exposed) results in GTP
hydrolysis. The MT cartoon (bottom right) shows an oversimplified representation of
a GTP cap as it first grows by tubulin addition and then shrinks by polymerization-coupled
GTP hydrolysis (here b-tubulin that is bound to GTP is shown in red and that bound
to GDP is shown in blue). Cryo-EM density map (EMDB-6349) and atomic model (PDB: 3JAK)
for an EB3-decorated MT bound to GTPgS. a-tubulin, b-tubulin and EB3 are colored green,
blue, and orange, respectively.
PI-014
Designed repeat proteins as templates for photoactive molecules and fluorescent nanoclusters
Sara H. Mejias1,2, Antonio Aires1,2, Javier López-Andarias3, Pierre Couleaud1,2, Begoña
Sot1,2, Carmen Atienza3, Nazario Martín1,3, Aitziber L. Cortajarena1,2
1IMDEA Nanoscience, c/Faraday, 9, Ciudad Universitaria de Cantoblanco 28049, 2CNB-CSIC-IMDEA
Nanociencia Associated Unit “Unidad de Nanobiotecnología”, 3Departamento de Química
Orgánica I, Facultad de Química, Universidad Complutense
Self-assembly of biological molecules into defined functional structures has a tremendous
potential in nanopatterning, and the design of novel bionanomaterials and functional
devices. Molecular self-assembly is a process by which complex three-dimensional structures
with specified functions are constructed from simple molecular building blocks. We
present first the study and characterization of the assembly properties of modular
repeat proteins, in particular designed consensus tetratricopeptide repeats (CTPRs),
and their application as building blocks in order to generate functional nanostructures
and biomaterials. CTPR proteins can be assembled into self-standing thin films,1 and
thin nanometer fibers in solution.2 In this work, we show the use of the designed
consensus repeat proteins as scaffolds to template: (1) photoactive organic molecules,
and (2) fluorescent nanoclusters. 1.We explore the potential of CTPR proteins to arrange
donor-acceptor pairs for electro-active materials. In particular, porphyrin rings
arranged by CTPRs in a defined distance and orientation for favoring face-to-face
orientation which should lead to an improvement in the optoelectronic properties.
Our results confirm the successful ability of CTPR proteins to be used as scaffold
for ordering organic chromophores, while preserving their structure. The unique self
assembly properties of CTPR scaffolds have been exploited to generate ordered conductive
films of the protein-porphyrin conjugates. These results open the door to fabricate
hybrid protein-based solid devices. 2.We show results on the ability of CTPR to encapsulate
and stabilize fluorescent gold nanoclusters. We investigated the influence of the
protein sequence in the final properties of the nanoclusters. The structural and functional
integrity of the protein template is critical for future applications of the protein-cluster
complexes. Therefore synthetic protocols that retain the protein structure and function
have been developed. As a proof of concept, a CTPR module with specific binding capabilities
has been successfully used to stabilize nano clusters.
References:
1. Grove TZ, Regan L, Cortajarena AL. Nanostructured functional films from engineered
repeat proteins. J R Soc Interface. 2013; 10(83):20130051.
2. Mejías SH, Sot B, Guantes R, Cortajarena AL. Controlled nanometric fibers of self-assembled
designed protein scaffolds. Nanoscale. 2014 Oct 7;6(19):10982-8. doi: 10.1039/c4nr01210k.
PI-015
Engineering of proteins to develop biomimetic hematite-based biohybrid materials
Greta Faccio1, Krisztina Schrantz2, Linda Thöny-Meyer1, Artur Braun2, Julian Ihssen1,
1Laboratory For Biointerfaces, Swiss Federal Laboratories For Materials Science and
Technology, CH, 2Laboratory For High Performance Ceramics, Swiss Federal Laboratories
For Materials Science and Technology, CH
Biohybrid photoelectrochemical cells have been developed by functionalizing the hematite
photoanode with the light-harvesting cyanobacterial protein C-phycocyanin (PC) yielding
a substantial enhancement of the photocurrent density. Photoelectrochemical cells
combining light-harvesting proteins and inorganic semiconductors have potential for
the use in artificial photosynthesis. In this work we present processing routes for
the functionalization of hematite photoanodes with PC, including in situ co-polymerization
of PC with enzymatically-produced melanin and using a recombinantly produced PC 2.
Moreover, recombinant forms of the light-harvesting protein C-phycocyanin from Synechocystis
sp. PCC6803 were engineered to carry a peptide with affinity for hematite. Similarly,
a bacterial laccase was engineered to acquire affinity for hematite. Results obtained
from the different approaches to hematite functionalization and the advantages offered
by protein engineering will be presented.
1 J. Ihssen, A. Braun, G. Faccio, K. Gajda-Schrantz, L. Thöny-Meyer, Light harvesting
proteins for solar fuel generation in bioengineered photoelectrochemical cells; Current
Protein and Peptide Science, Accepted for publication.
2 Faccio G, Schrantz K, Ihssen J, Boudoire F, Hu Y, Mun BS, Bora DK, Thöny-Meyer L,
Braun A Charge transfer between photosynthetic proteins and hematite in bio-hybrid
photoelectrodes for solar water splitting cells, Nano Convergence, Accepted Nov 2014.
PI-016
Correcting free energy expressions for thermal motion
Martin Goethe1, Ignacio Fita2, J. Miguel Rubi1
1Department of Fundamental Physics, University of Barcelona, 2Molecular Biology Institute
of Barcelona (ibmb-CSIC)
Minimizing a suitable free energy expression is arguably the most common approach
in (ab initio) protein structure prediction. The achieved accuracy depends crucially
on the quality of the free energy expression in use. Here, we present corrections
to existing free energy expressions which arise from the thermal motion of the protein.
We (i) devise a term accounting for the vibrational entropy of the protein, and (ii)
correct existing potentials for ’thermal smoothing’. (i) Vibrational entropy is almost
always neglected in free energy expressions as its consideration is difficult. This
practice, however, may lead to incorrect output because distinct conformations of
a protein can contain very different amount of vibrational entropy, as we show for
the chicken villin headpiece explicitly [1]. For considering vibrational entropy,
we suggest a knowledge based approach where typical fluctuation and correlation patterns
are extracted from known proteins and then applied to new targets. (ii) At ambient
conditions, time-averaged potentials of proteins are considerably smoothened due to
thermal motion where the strength of this effect varies strongly between atoms. Distinguishing
these inhomogeneities by introducing new atom species regarding their locale environment
can therefore increase the precision of time-averaged potentials [2]. * Department
of Fundamental Physics, University of Barcelona ** Molecular Biology Institute of
Barcelona (ibmb-CSIC)
[1] M. Goethe, I. Fita, and J.M. Rubi, Vibrational Entropy of a Protein: Large Differences
between Distinct Conformations, J. Chem. Theory Comput. 11, 351 (2015).
[2] M. Goethe, I. Fita, and J.M. Rubi, in preparation.
PI-017
Tertiary structural propensities reveal fundamental sequence/structure relationships
Fan Zheng1, Craig Mackenzie2, Jian Zhang3, Gevorg Grigoryan1,3
1Department of Biological Sciences, Dartmouth College, 2Institute For Quantitative
Biomedical Sciences, Dartmouth College, 3Department of Computer Science, Dartmouth
College
Extraction of general principles from the continually growing Protein Data Bank (PDB)
has been a significant driving force in our understanding of protein structure. Atomistic
or residue-level statistical potentials, secondary-structural propensities, and geometric
preferences for hydrogen bonding are among the classical insights that arose from
observations in the PDB. Given the magnitude of structural data available today, it
is likely that many quantitative generalizations remain to be made. Here we hypothesize
that the PDB contains valuable quantitative information on the level of local tertiary
structural motifs (TERMs), with TERM statistics reflecting fundamental relationships
between sequence and structure. We define a TERM to be the structural fragment that
captures the local secondary and tertiary environments of a given residue, and put
our hypothesis through a series of rigorous tests. First, we show that by breaking
a protein structure into its constituent TERMs, and querying the PDB to characterize
the natural ensemble around each, we can estimate the compatibility of the structure
with a given amino-acid sequence through a metric we term “structure score.” Considering
submissions from recent Critical Assessment of Structure Prediction (CASP) experiments,
we find a strong correlation (R = 0.69) between structure score and model accuracy,
with poorly predicted regions readily identifiable. This performance exceeds that
of leading atomistic statistical energy functions. Next, we show that by considering
the TERMs of a structure that are affected by a given mutation, and mining the PDB
to characterize sequence statistics associated with each, we are able to predict mutational
free energies on par with or better than far more sophisticated atomistic energy functions.
Finally, we ask whether TERM statistics are sufficient to enable the design of proteins
de-novo. We demonstrate that given a native backbone conformation, TERM considerations
alone with no input from molecular mechanics correctly predict roughly the same fraction
of amino acids from the corresponding native sequence as state-of-the-art computational
protein design methods. Knowledge-based energy functions have already put PDB statistics
to good use by parsing structural environments into geometric descriptors, generally
assuming their conditional independence. Our results suggest that it may now be possible
to instead consider local structural environments in their entirety, asking questions
about them directly. If this is the case, then the PDB is an even larger treasure
trove of information than it has been generally known to be, and methods of mining
it for TERM-based statistics should present opportunities for advances in structure
prediction and protein design.
PI-018
De novo design of an ideal TIM-barrel scaffold
Po-Ssu Huang1,2, Kaspar Feldmeier3, Fabio Parmeggiani1,2, D. Alejandro Fernandez Velasco4,
Birte Höcker3, David Baker1,2,5
1Department of Biochemistry, University of Washington, 2Institute for Protein Design,
University of Washington, 3Max Planck Institute for Developmental Biology, 4Facultad
de Medicina, Universidad Nacional Autónoma de México, Ciudad Universita, 5Howard Hughes
Medical Institute, University of Washington
Comprehensive understanding of a protein fold is intertwined with successful design.
Recent advances in designing de novo structures have shown that proteins can be designed
for a few globular and helical folds. However, designing all-β structures and barrels
remains challenging because loops and intricate long range interactions that are important
in these topologies are difficult to control. For designing novel catalysts, the (α/β)8
-barrel (or TIM-barrel) fold is one of the most important examples, for it is the
most common topology for enzymes. For almost 30 year, attempts in designing de novo
TIM barrel structures have all resulted in poorly folded proteins. Here we describe
the successful design of a 4-fold symmetrical (α/β)8 barrel directly from geometrical
and chemical principles. 22 designed variants with a wide range of stabilities from
being molten globules to cooperatively folded proteins were experimentally characterized,
and the results revealed the importance of sidechain-backbone hydrogen bonding for
defining the characteristic α/β-barrel. The 184 residue TIM barrel structure is among
the smallest TIM-barrels and has a fully-reversible melting temperature of 88°C. The
X-ray crystal structure shows atomic-level agreement with the design model. Despite
this structural similarity, PSI-BLAST searches do not identify sequence similarities
to known TIM-barrel proteins. More sensitive profile-profile searches suggest that
the design is sufficiently distant from other native TIM-barrel superfamilies to be
in a superfamily of its own, further implying that Nature has only sampled a subset
of the sequence space available to the TIM-barrel fold. The ability to de novo design
TIM-barrels opens new possibilities for custom-made enzymes.
PI-019
Directed evolution of fluorescent protein function
Felix Vietmeyer1, Premashis Manna1,2, Kevin Dean3, Amy Palmer3, Ralph Jimenez1,2
1JILA, University of Colorado and NIST, 2Dept. of Chemistry & Biochemistry, University
of Colorado, 3University of Texas Southwestern Medical Center, 4BioFrontiers Institute,
University of Colorado
Creation of new molecular sensors and actuators based on fluorescent proteins relies
on methods for identifying complex photophysical phenotypes and subsequently performing
separations on cell populations. We developed a microfluidic flow cytometry approach
tailored to interrogating the performance of genetically-encoded fluorophores and
present the results of studies employing this technology. The system screens cell-based
libraries on the basis of multiple photophysical parameters relevant to imaging, including
brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence
quantum yield) at a rate of up to 180 cells/sec. In a first generation of experiments,
molecular dynamics-guided design was used to create a library of mCherry mutants that
was screened with this system, resulting in the identification of a variant with a
higher stability β-barrel and improved photostability but with a decreased brightness
due to reduction in the fluorescence quantum yield. To avoid inadvertent decreases
in this important performance criterion, subsequent rounds of selection were performed
on the basis of both photostability and excited-state lifetime as sorting criteria.
In these second generation selections, mutations were designed to target pathways
of oxygen access through the bottom of the β-barrel in addition to a position that
directly interacts with the chromophore. Furthermore, subsequent rounds of screening
were used to improve folding and maturation. The multiparameter sort identified multiple
clones with up to 8-fold improved photostability and up to double the excited-state
lifetime of the parent mCherry fluorescent protein. The best mutant we identified
produces one order of magnitude more photons before photobleaching compared to mCherry,
at excitation conditions characteristic of confocal fluorescence microscopy. Our results
demonstrate the utility of combining molecular-dynamics-guided library design with
technology for photophysics-based selections. We anticipate that the new fluorescent
proteins obtained in this work will find use in low-copy-number and long-duration
imaging live cell imaging applications in cell-lines created by genomic editing techniques.
PI-020
Targeted protein degradation achieved through a combination of degrons from yeast
and mammalian ornithine decarboxylase
Rushikesh Joshi1, Ratna Prabha C.1
1The Maharaja Sayajirao University of Baroda
Targeted protein degradation achieved through a combination of degrons from yeast
and mammalian ornithine decarboxylase Targeting the over accumulated protein in the
cell for degradation using specific degrons is an emerging research area. The degradation
of the vast majority of cellular proteins is targeted by the ubiquitin-proteasome
pathway. But in the case of ubiquitin independent protein degradation, ODC/AZ system
is more effective in achieving targeted protein degradation than other types of degradation
1. Ornithine decarboxylase (ODC) is key regulatory enzyme in the biosynthesis of polyamines.
The protein has two domains namely, N terminal α/β barrel domain and C-terminal β-sheet
domain. Degradation of ODC is mediated by polyamine inducible protein, antizyme (AZ).
Antizyme interacts with ODC on N-terminal region, which results in degradation of
ODC by proteasomes. In mammalian ODC the C-terminal has an unstructured tail of 37
residues, which pulls ODC into proteasome for degradation. It was reported earlier
by Coffino’s group that the unstructured tail acts as a degron in chimeric fusion
with GFP 2. In yeast, same function is achieved by N-terminal 44 residues 3. Present
study focuses on accomplishing targeted protein degradation in Saccharomyces cerevisiae
by adding these two degradation signals or degrons of yeast ODC and mammalian ODC
as tags to a reporter protein. We have selected two degrons namely, N terminal α/β
barrel domain of yeast ODC and C-terminal 37 residues of mouse ODC and grafted them
to N and C- terminus of the reporter protein yEGFP. Degradation of yEGFP and yEGFP
fusion with degrons of ODC (degron-yEGFP) were monitored by western blot using anti-GFP
antibody and fluorescence spectroscopy. Initially, the amount of degron-yEGFP fusion
protein was very low compared to control yEGFP. It means that the chimeric protein
underwent rapid degradation in the cells. After inhibition of proteasome, increase
in the level of degron-yEGFP was observed, confirming that the degrons cause rapid
degradation of reporter protein through proteasome. Earlier, we have also tagged ubiquitin
from yeast with last 37 residues of mODC and observed enhanced degradation of ubiquitin
in Saccharomyces cerevisiae. Therefore, both the degrons of ODC alone and in combination
are capable of decreasing stability of reporter protein in the cells. However, the
combination of degrons is more effective than either of them in isolation.
References:
1 Matsuzawa S. et al. 2005, Proc Natl Acad Sci USA 102, 14982-14987.
2 Zhang M. et al. 2003, Embo J 22, 1488-1496.
3 Godderz D. et al. 2011, J Mol Biol 407, 354-367.
Corresponding author: Department of Biochemistry, Faculty of Science, The Maharaja
Sayajirao University of Baroda, Vadodara – 390002, India. e-mail: chivukula_r@yahoo.com;
Mobile: +91-9327201349
PI-021
Short peptides self-assemble in the presence of metals to produce catalytic amyloids
Caroline Rufo1, Yurii Moroz1, Olesia Moroz1, Olga Makhlynets1, Pallavi Gosavi1, Jan
Stöhr2, Tyler Smith1, Xioazhen Hu3, William DeGrado3, Ivan Korendovych1
1Syracuse University, 2Institute for Neurodegenerative Diseases and Department of
Neurology, UCSF, 3Department of Pharmaceutical Chemistry, UCSF
Enzymes fold into unique three-dimensional structures, which underlie their remarkable
catalytic properties. The requirement that they be stably folded is a likely factor
that contributes to their relatively large size (> 10,000 Dalton). However, much shorter
peptides can achieve well-defined conformations through the formation of amyloid fibrils.
To test whether short amyloid-forming peptides might in fact be capable of enzyme-like
catalysis, we designed a series of 7-residue peptides that act as Zn2+-dependent esterases.
Zn2+ helps stabilize the fibril formation, while also acting as a cofactor to catalyze
acyl ester hydrolysis. The fibril activity is on par with the most active to date
zinc-protein complex. Such remarkable efficiency is due to the small size of the active
unit (likely a dimer of 7-residue peptides), while the protein is at least 15-fold
larger in molecular weight. The observed catalytic activity is not limited to ester
hydrolysis. We have designed copper binding peptides that are capable oxygen activation.
These results indicate that prion-like fibrils are able to not only catalyze their
own formation – they also can catalyze chemical reactions. Thus, they might have served
as intermediates in the evolution of modern-day metalloenzymes. These results also
have implications for the design of self-assembling nanostructured catalysts including
ones containing a variety of biological and nonbiological metal ions.
PI-022
Rational design of the cold active subtilisin-like serine protease VPR with improved
catalytic properties and thermal stability
Kristinn Oskarsson1, Sigridur Thorbjarnardottir2, Magnus Kristjansson1
1Science Institute, University of Iceland, Department of Biochemistry, 2Institute
of Biology, University of Iceland
Rational design of the cold active subtilisin-like serine protease VPR with improved
catalytic properties and thermal stability. Kristinn R. Óskarsson1, Sigridur H. Thorbjarnardóttir2,
Magnús M. Kristjánsson1. 1Science Institute, University of Iceland, Department of
Biochemistry, Reykjavík, Iceland. 2University of Iceland, Institute of Biology. mmk@hi.is
On the basis of research done on the subtilisin-like serine proteinase VPR, from a
psychrophilic Vibrio species and its thermophilic structural homologue, aqualysin
I (AQUI) from Thermus aquaticus, we set out to design a mutant of VPR which would
be more thermostable, but would retain the high catalytic activity of the wild type
enzyme. Our starting protein template was a previously stabilized mutant containing
two inserted proline residues close to the N-terminus of VPR (N3P/I5P). This VPR_N3P/I5P
mutant was shown to have a significantly increased thermal stability but displayed
a concomitant tenfold loss of catalytic efficiency. From our previous studies we selected
two mutations, one which increased catalytic activity (Q142K) of the enzyme significantly
and another which stabilized the protein against thermal denaturation (N15D). The
N15D mutation had been shown to introduce a salt bridge into the structure of the
cold adapted proteinase, yielding higher stability but without negative effects on
activity. The Q142K exchange had been shown to double the turnover number (kcat) to
that of the wild type enzyme. Insertions of these selected mutations into the VPR_N3P/I5P
mutant were according to predictions; the Q142K increased the kcat tenfold, and the
N15D mutation increased the thermal stability. In the combination mutant, VPR_N3P/I5P/N15D/Q142K,
thermal stability was increased by 8°C and 10°C, in terms of Tm and T50%, respectively.
Furthermore, the catalytic activity of the mutant was somewhat higher than that of
the wild type enzyme.
PI-023
Critical peptide stretches may not serve as faithful experimental mimics for protein
amyloidogenesis
Bishwajit Kundu1, Dushyant Garg1
1Kusuma School of Biological Sciences, IIT Delhi
Certain amino acid stretches are considered critical to trigger the amyloidogenesis
in a protein. These peptide stretches are often synthetically produced to serve as
experimental mimics for studying amyloidogenesis of the parent protein. Here we provide
evidence that such simple extrapolation may be misleading. We studied the amyloidogenesis
of full length bovine carbonic anhydrase II (BCAII) and compared it with those formed
by its critical amyloidogenic peptide stretch 201-227 (PepB). Under similar solution
conditions and initial monomeric concentrations, we found that while amyloid formation
by BCAII followed aggregation kinetics dominated by surface-catalyzed secondary nucleation,
PepB followed classical nucleation-dependent pathway. The AFM images showed that BCAII
forms short, thick and branched fibrils, whereas PepB formed thin, long and unbranched
fibrils. ATR-FTIR revealed parallel arrangement of cross β sheet in BCAII amyloids,
while PepB arranged into antiparallel β sheets. Amyloids formed by BCAII were unable
to seed the fibrillation of PepB and vice versa. Even the intermediates formed during
lag phase revealed contrasting FTIR, far UV CD signature, hydrophobicity and morphology.
We propose that for any polypeptide, the sequences flanking a critical region are
equally effective in modulating the initial nucleation events, generating prefibrillar
and finally fibrillar species with contrasting characteristic. The results have been
discussed in light of amyloid polymorphism and its importance in the design of therapeutic
strategies targeting such toxic regions.
PI-024
A systematic exploration of protein uptake and trafficking into intracellular compartments
Aksana Labokha1, Ralph Minter1
1Antibody Discovery & Protein Engineering dpt, MedImmune
All approved biological drugs target extracellular proteins and not the majority of
the expressed human genome, which resides within intracellular compartments. Included
in the latter category are many important, disease-relevant targets which cannot be
easily addressed by small molecule approaches, such as the oncology targets c-Myc
and K-Ras. Although bacteria and viruses have evolved strategies to deliver biological
material to the cell cytoplasm and nucleus, our ability to engineer recombinant proteins
to replicate this is somewhat limited by (i) our nascent understanding of protein
uptake and trafficking pathways and (ii) the ability to easily quantify cell delivery
to the cytoplasm and cellular organelles. The aim of my project is to address these
challenges by developing an effective assay for cytoplasmic uptake and then using
it to measure the delivery efficiency of recombinant proteins which mimic natural
delivery strategies e.g. cell penetrating peptides fusion, exotoxin mimics, and supercharged
proteins (proteins with high surface charge which can enter cells). I also intend
to explore the influence of the Rab superfamily, which are the master regulators of
protein trafficking, to influence and control both the kinetics and final subcellular
destination of exogenous proteins.
PI-025
Protein engineering: what’s next?
Maria Fatima Lucas1,2, Víctor Guallar1,3
1Joint BSC-CRG-IRB Research Program in Computational Biology, 2Anaxomics Biotech,
3ICREA
With the growing industrial need for engineering enzymes for the deconstruction and
transformation of plant biomass in biorefineries, there is a want for the development
of new approaches for designing special purpose biocatalysts. Techniques, such as
directed evolution, which mimic the natural selection process by evolving proteins
towards the improvement of a given property, have unquestionably demonstrated their
value and are routinely used in large industrial companies. Nevertheless, the brute
force employed in these methods, could significantly gain from an all-atom description
of the underlying catalytic mechanisms, to center the efforts on more limited areas
of the protein. In the last years, we have developed computational tools, which combine
the electronic structure description of QM/MM methods with the potential to model
long time scale processes of PELE,1 to study the details of a variety of reactions.
Examples, which will be discussed, include rationalizing the selective oxyfunctionalization
of steroids using fungal enzymes2 and the study of the effect of point mutations on
the oxidation efficiency of laccases.3 These methods have shown their potential not
only at the descriptive level but, more importantly, through their high predictive
capability that opens many opportunities for their use in biotechnology. In this talk,
we will show how recent advances in in silico approaches are setting new grounds for
future computer guided directed evolution.
This work was done in collaboration with: Instituto de Recursos Naturales y Agrobiología
de Sevilla, CSIC; Novozymes A/S; JenaBios GmbH; TU Dresden; Centro de Investigaciones
Biológicas, CSIC and was funded by the INDOX (KBBE-2013-7-613549) European Project.
1-Borrelli K., et al. PELE: Protein energy landscape exploration. A novel Monte Carlo
based technique. J. Chem. Theory Comp. 1, pp. 1304. 2005.
2-Babot, B., et al. Steroid hydroxylation by basidiomycete peroxygenases: A combined
experimental and computational study. Applied and Environmental Microbiology. 2015.
Accepted manuscript posted online doi: 10.1128/AEM.00660-15
3-Monza, E., et al. Insights into Laccase Engineering from Molecular Simulations:
Toward a Binding-Focused Strategy. Journal Physical Chemical Letters. 6, pp. 1447
- 1453. 2015.
PI-026
Bottom-up construction of a synthetic carboxysome
Shiksha Mantri1, Raphael Frey1, Marco Rocca1, Eita Sasaki1, Donald Hilvert1
1ETH Zurich, Switzerland, 2ETH Zurich, Switzerland, 3ETH Zurich, Switzerland, 4ETH
Zurich, Switzerland, 5ETH Zurich
Several orthogonal bioreactions take place simultaneously within membrane bound organelles
in eukaryotes and proteinaceous microcompartments in bacteria. These subcellular structures
contain sets of enzymes co-involved in metabolic pathways. Towards the goal of creating
artificial protein microreactors, we seek to develop an artificial organelle that
emulates the metabolic activity of the carbon fixating organelle of autotrophic bacteria,
the carboxysome. Here, we show that the two key carboxysomal enzymes, ribulose-1,5-bisphosphate
carboxylase/oxygenase (RuBisCO) and carbonic anhydrase (CA), can be efficiently co-encapsulated
using our previously reported encapsulation system which is based on a bacterial capsid
formed from the protein lumazine synthase (AaLS-13). Our preliminary results suggest
that the enzymes can act in tandem and that the co-encapsulation of CA with RuBisCO
in the capsid is necessary for enhanced RuBisCO activity in vitro. We attribute this
observation to the high local concentrations of the RuBisCO substrate, CO2, produced
by CA within the capsid. We are developing a theoretical model of a minimal carboxysome
using the kinetic rate constants of our RuBisCO and CA variants and AaLS-13 as the
shell to complement these experiments. Next, we will incorporate our minimal carboxysome
within an expression host such as E.coli, opening up the possibility of further optimization
through directed evolution.
PI-027
CXCL10 engineering: novel insights into glycan interactions
Michael Nagele1, Martha Gschwandtner1, Patrick Sorger1, Andreas J. Kungl1
1Institute of Pharmaceutical Sciences, University of Graz, Universitaetsplatz 1
In the past targeting and engineering of chemokines has led to several interesting
drug candidates. [1] Amongst them, Met-RANTES, a Met-CCL5 with high G protein-coupled
receptor (GPCR) affinity but no subsequent signal transduction, as well as mutants
addressing the interaction with the so-called glycosaminoglycans (GAGs) seem to be
the most promising candidates. Both, GAG knockout as well as GAG affinity matured
chemokine isoforms have been considered as anti-inflammatory drug candidates, out
of which an IL-8 mutant with 5 modifications reached clinical phase 1 where it was
profiled for acute neutrophil-related exacerbation in COPD.[2] CXCL10 (IP-10) is a
proinflammatory chemokine released by various cells following stimulation by interferon
γ (IFN-γ). It is therefore considered as a late chemokine being responsible for the
attraction of different lymphocytes. [3] Any therapeutic indication is consequently
related to chronic and multiple applications. We have therefore engineered CXCL10
very conservatively at positions to ultimately generate dominant-negative mutants
with a mildly improved GAG-binding affinity and an entire knock off GPCR activity.
The first steps of our engineering approach were in silico modelling of the mutants
and the establishment of a suitable upstream- and downstream-processing protocol.
Next we generated a fluorescently engineered CXCL10 variant for our fluorescence-based
affinity studies which was subjected to biocomparability investigations relative to
the native, non-fluorescent protein. Compared to the wild type, the fluorescently
engineered mutant exhibited similar biological, chemotactic and GAG-binding properties.
Next we started to produce sufficient amounts of the members of our nascent mutant
library which were tested with respect to their biophysically behavior as well as
to their knocked out chemotactic potency on cells. These experiments included gel
electrophoresis and Western Blot analysis to determine identity and purity; Circular
Dichroism (CD) and chaotrope-induced unfolding to approximate structure; Isothermal
Fluorescence Titration (IFT); Surface Plasmon Resonance (SPR) and Isothermal Titration
Calorimetry (ITC) to quantify GAG-binding affinity and Boyden Chamber experiments
to determine the chemotactic activity. Our results show that we are able to tune the
GAG binding strength along with the GPCR activity of human CXCL10 which could lead
to therapeutic applications in the future.
[1] Proudfoot AE, Power C, Schwarz MK. Anti-chemokine small molecule drugs: a promising
future? Expert Opin. Investig. Drugs 19(3):345-355.2010
[2] Falsone A, Wabitsch V, Geretti E, Potzinger H, Gerlza T, Robinson J, Adage T,
Teixeira M, Kungl A. Designing CXCL8-based decoy proteins with strong anti-inflammatory
activity in vivo. Biosci Rep.2013;33(5):e00068.2013
[3] Campanella G, Lee E, Sun J, Luster A. CXCR3 and Heparin Binding Sites of the Chemokine
IP-10 (CXCL10). J.Biol.Chem.2003, 278:17066-17074.2003
PI-028
Creating large covalently circularized nanodiscs and their application in studying
viral entry and genome translocation
Mahmoud Nasr1, Mike Strauss1, James Hogle1, Gerhard Wagner1
1Dep. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School
Nanodiscs are composed of a nanometer-sized phospholipid bilayer encircled by two
α helical, amphipathic membrane scaffold proteins (MSPs). These particles provide
a unique detergent free lipid bilayer model enabling biochemical and biophysical characterization
of membrane proteins in a physiologically relevant medium. Previously, the largest
diameter reported of a nanodisc assembled using MSPs was about 16-17 nm. Here we present
a method to create large nanodiscs (up to 80nm in diameter) assembled with covalently
circularized MSPs (cMSP). We can observe the homogeneity in nanodiscs diameter as
a narrow distribution using negative-stain EM. Using our method, we have created 50
nm nanodiscs and used them to study poliovirus (∼35 nm diameter) entry and RNA translocation.
A 50 nm nanodisc is sufficiently large to accommodate multiple copies of the CD155
receptor (also known as the poliovirus receptor), and has enough surface area to act
as a surrogate membrane for the RNA translocation complex during viral uncoating.
The 50 nm nanodiscs functionalized with the His-tagged ecto-domain of poliovirus receptor,
CD155, were generated by adding lipids derivatized with a NTA nickel-chelating head
group to the lipid mixture during nanodisc assembly. CD155 receptor was added to the
already assembled nanodiscs and incubated for 30 minutes at room temperature. The
receptor-decorated nanodisc complex was purified by size exclusion chromatography.
The purified complex was then incubated with poliovirus for 5 minute at 4ºC, and then
heated to 37ºC for 15 minutes to initiate receptor-mediated viral uncoating. Virus
binding to nanodisc-CD155 complex and subsequent insertion of viral components into
and across the membrane were confirmed by negative-stain electron microscopy (Figure
1c). To obtain a high-resolution structure for the RNA translocation complex we conducted
single-particle cryo-EM studies using a Polara F30 microscope. Unlike liposomes, generating
a reconstruction of samples containing nanodiscs is less complicated since the nanodiscs
are more homogenous in size, and allow for thinner ice. Also, the viral RNA can be
visualized more easily. The method for making large nanodiscs as well as the negative
stain and cryo-EM data will be will be presented and discussed.
Figure 1.
Poliovirus- membrane fusion studies using large nanodisc. (a) Negative stain EM of
50 nm nanodiscs plus poliovirus (control) shows no bridging or fusion. (b) Outline
of the procedure for initiating poliovirus bridging and fusion with nanodiscs modified
with CD155. CD155 is also known as the poliovirus receptor. (c) Individual viruses
tethered to nanodiscs.
PI-030
Parametric design of alpha-helical barrels and pore-like assemblies with very high
thermodynamic stabilities
Gustav Oberdorfer1,2,7, Po-Ssu Huang1,7, Chunfu Xu1,7, Verena Kohler2, Xue Y. Pei3,
Brent L. Nannenga4, Joseph M. Rogers5, Tamir Gonen4, Karl Gruber2, David Baker1,6,7
1Department of Biochemistry, University of Washington, 2Institute of Molecular Biosciences,
University of Graz, 3Department of Biochemistry, University of Cambridge, 4Janelia
Research Campus, Howard Hughes Medical Institute, 5Department of Chemistry, University
of Cambridge, 6Howard Hughes Medical Institute, University of Washington, 7Institute
for Protein Design, University of Washington
Computational design of novel protein structures and enzymes with new functions is
a promising tool to create superior biological materials with tailor-made properties,
new pharmaceuticals, complex fine chemicals or renewable fuels. It also challenges
our understanding of protein folding, protein evolution, molecular recognition and
catalysis. Here we present a procedure for designing proteins with backbones produced
by varying the parameters in the Crick coiled-coil generating equations [1]. Combinatorial
design calculations using the software suite Rosetta identify low energy sequences
for alternative helix supercoil arrangements. After that, loop modeling is applied
to connect the designs with lowest energy. The extent to which the designed sequences
encode the designed structures is evaluated using large-scale structure prediction
calculations, as well as symmetric and asymmetric protein-protein docking calculations.
Subsequently, synthetic genes are generated for sequences that converge strongly on
the designed structure for experimental characterization. We applied this approach
to monomeric three and four helical bundle structures as well as a pentameric five-helix
bundle structure using idealized coiled-coil geometries [2]. Recently we expanded
this approach to higher complexity backbones, which resulted in the de-novo design
of monomeric, antiparallel six-helix bundles with untwisted, left- and right-handed
geometries. Circular Dichroism (CD), Size-exclusion coupled Multi-Angle Light Scattering
measurements (SEC-MALS), negative stain electron micrographs (EM) and Small Angle
X-ray Scattering (SAXS) of these designs suggest that they indeed form the designed
structures. In addition, we used Rosetta protein-protein interface design functionality
to computationally design oligomers out of our previously published three and four
helix bundle structures to generate self-assembling pore-like structures with the
potential use as channels or transporters. Again, experimental validation of these
designs by CD, SEC-MALS, EM and SAXS show that the designs are correct. We are currently
undertaking further structural investigation of all these designs by X-ray crystallography.
The designs described above can act as templates for protein or small molecule binding,
holding a catalytic machinery or for scaffolding enzymes in reaction cascades. Some
of these applications are currently under investigation, including a self-sufficient
redox system employing two copper-centers, binding of heme-moieties as a prosthetic
group and tailoring the pore-like geometries to be used in nanopore sequencing.
[1] F. H. Crick. (1953) The Fourier Transform of a Coiled Coil, Acta Cryst., 6: 685
[2] *Huang, P-S., *Oberdorfer, G., *Xu, C., et al. (2014) High thermodynamic stability
of parametrically designed helical bundles. Science, 24 October 2014: 481-485
*equal contribution
PI-031
Leucine Zipper fused Fab; Enhancement of active Fab formation in E. coli in vitro
and in vivo expression systems
Teruyo Ojima-Kato1, 2, Kansuke Fukui2, Takaaki Kojima2, Hideo Nakano2
1Aichi Science and Technology Foundation, 2Nagoya University
Background: Recombinant monoclonal antibodies (mAbs) are one of the essential tools
in biotechnology. The smaller fragments of antibodies such as fragment of antigen
binding (Fab) or single chain variable fragment (scFv) are favorable form for the
production in microorganisms. These have been produced in periplasmic space in E.coli,
however, the productivity is still limited. On the other hand, cytoplasmic production
of these molecules could enlarge their productivity greatly, however, often results
in insoluble aggregates. In particular, soluble expression of Fab is challenging because
intermolecular disulfide bond between heavy chain and light chain is hardly formed
in the E.coli cytoplasm. Purpose: To achieve functional Fab production in E. coli
in vitro and in vivo expression system, peptides segment pairs of Leucine zipper that
dimerize in parallel were fused to the C-termini of Fab. Results: Mouse anti E. coli
O157 mAb obtained from Hybridoma and rabbit anti Listeria monocytogenes mAb generated
by single B cell RT-PCR were used as model. These mAbs were not produced in E. coli
as active Fab. Leucine zipper (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused
to the C-terminal of heavy chain (VH-CH1) and light chain (VL-CL), respectively, to
accelerate the association of the heavy chain and light chain of Fab. The modified
Fabs were produced in either E. coli in vitro or in vivo expression systems as active
proteins. SDS-PAGE analysis of the purified Fab-Leucine zipper conjugates showed absence
of disulfide bond between heavy and light chains, indicating they were connected via
leucine zipper interaction. These leucine zipper fused Fab(s) had significant binding
and specificity toward antigen in ELISA. Conclusions: Leucine zipper fusion to Fab
greatly enhanced assembly of heavy chain and light chain to form active Fab.
PI-032
The road not taken: Exploring repeat protein architectures by computational design
Fabio Parmeggiani1, Po-Ssu Huang1, TJ Brunette1, Damian Ekiert2, Gira Bhabha2, Susan
Tsutakawa3, Greg Hura3, John Tainer3, David Baker1
1University of Washington, 2University of California, San Francisco, 3Lawrence Berkeley
National Laboratory
Repeat proteins are an example of how evolution proceeds by building on existing structures
and functions, but also a source of modular protein scaffolds for molecular recognition
and biomaterials. However, it is unclear whether the limited number of folds and families
that we know today is the result of the intrinsic limitations of polypeptide chains
or the consequence of the path followed by evolution. We explored this hypothesis
by computational design of repeat proteins based on modular units formed by two alpha
helices and two loops of variable lengths, without relying on information from available
repeat protein families. The automated sampling of the conformational space resulted
in a large number of architectures from which 83 de novo designs were selected for
experimental characterization. 66% of the proteins were stable up to 95°C and monodisperse
and 42 designs were structurally validated by small angle X-ray scattering. Crystal
structures were solved for 15 of them, with root mean square deviation from the models
between 0.7Å and 2.5Å. The designs differ from known proteins both at the sequence
and structure levels and cover a broader range of geometries than observed in naturally
occurring repeat protein families, indicating that existing architectures represent
only a small fraction of what can be achieved. Our results show that it is possible
to expand the range of repeat protein architectures beyond the naturally occurring
families, and that computational design can provide new scaffolds and enable the design
of proteins tailored for specific applications.
PI-033
Design and characterisation of a synthetic serpin with novel folding properties
Benjamin Porebski1, Shani Keleher1, Adrian Nickson2, Emilia Marijanovic1, Mary Pearce1,
Natalie Borg1, James Whisstock1, Stephen Bottomley1, Sheena McGowan1, Ashley Buckle1
1Department of Biochemistry and Molecular Biology, Monash University, 2Department
of Chemistry, University of Cambridge
The serpin family of proteins consists of over 1500 members, all with a highly conserved
native structure that is metastable (1). Serpins use this metastability to control
the activity of proteases, via a specific inhibitory process. The serpin binds to
its target protease through specific residues within the reactive centre loop, the
protease cleaves the loop and results in a large conformational change causing the
protease to become distorted and catalytically inactive whilst the serpin becomes
much more stable (1, 2, 3). The metastable nature of AAT is therefore required to
facilitate the rapid and gross conformational changes required for its inhibitory
function (2, 3). Several disease-causing mutants of AAT have been identified, the
most common of them being the Z-variant (4). The Z-variant has an increased propensity
to polymerize in the endoplasmic reticulum of hepatocytes leading to cell death and
liver damage (4). During the past fifteen years, many groups have unsuccessfully screened
a number of serpins and a vast range of solution conditions to identify a combination
of serpin and conditions that will enable the folding reaction of a serpin to be characterized.
We have now taken an alternative approach and designed a synthetic “model” serpin
that folds reversibly to its native state. In order to do this, we used a consensus
design approach, analysing a sequence alignment of 212 serpin sequences and determining
the prevalent amino acid residue at each position, we termed this serpin conserpin
(consensus serpin). Here we present the structural, biophysical and functional characterisation
of conserpin. Combined crystallographic and folding studies reveal the characteristics
of conserpin that likely dictate its unique stability and folding behaviour, whilst
retaining activity as a serine protease inhibitor.
References:
1. Law, R. H. P., Zhang, Q., McGowan, S., Buckle, A. M., Silverman, G. A., Wong, W.,
et al. (2006). An overview of the serpin superfamily. Genome Biology, 7(5), 216.
2. Huntington JA, Read RJ, & Carrell RW (2000) Structure of a serpin-protease complex
shows inhibition by deformation. Nature 407(6806):923-926.
3. Tew DJ & Bottomley SP (2001) Intrinsic fluorescence changes and rapid kinetics
of proteinase deformation during serpin inhibition. FEBS Letters 494(1-2):30-33.
4. Lomas DA, Evans DL, Finch JT, & Carrell RW (1992) The mechanism of Z alpha 1-antitrypsin
accumulation in the liver. Nature 357(6379):605-607.
PI-034
Computational design of shape-optimized leucine-rich repeat proteins
Sebastian Rämisch1, Ulrich Weininger2, Jonas Martinsson1, Mikael Akke2, Ingemar André1
1Department for Biochemistry & Structural Biology, Lund University, 2Department for
Biophysical Chemistry, Lund University
The development of enhanced protein binding scaffolds is a key for engineering protein
inhibitors and biosensors with advanced characteristics. Utilizing the structural
variability and designability of repeat proteins offers a means for designing protein
binders where the overall shape is customized to optimally match a target molecule.
We developed a computational protocol for the design of repeat proteins with a predefined
geometry. By combining sequence optimization of existing repeats and de novo design
of capping structures, we designed leucine-rich repeat (LRR) proteins where the building
blocks assemble into a novel structure. The suggested design procedure was validated
by engineering an artificial donut-like ring structure, which is constructed from
ten self-compatible repeats. Characterization of several designed constructs further
suggests that buried cysteines play a central role for stability and folding cooperativity
in certain LRR proteins. This effect could provide a means for selectively stabilizing
or destabilizing specific parts of an LRR-based protein binder. The computational
procedure may now be employed to develop repeat proteins with various geometrical
shapes for applications where greater control of the interface geometry is desired.
PI-035
Engineering APOBEC3G enzymes for altered specificity and processivity
Louis Scott1, Muhammad Razif1, Aleksandra Filipovska1,2, Oliver Rackham1,2
1Harry Perkins institute of Medical Research, 2School of Chemistry and Biochemistry,
The University of Western Australia
APOBEC3G (A3G) is a host-encoded protein involved in the defense against HIV-1 and
other retroviral infections. A3G is a cytidine deaminase with a 3’ to 5’ processive
nature, causing targeted C to T mutations along a DNA strand. The catalytic and processive
activity of A3G leads to the hypermutation of nascent retroviral cDNA, resulting in
premature termination codons and dysfunctional proteins. Ultimately, the action of
A3G inhibits viral replication. The ability of A3G to jump and slide along a DNA strand,
deaminating at targeted sequences, makes it an interesting candidate for protein engineering.
Engineered A3G enzymes for increased activity, altered specificity, and altered processivity
are attractive options for expanding the DNA modifying enzyme toolbox. Mutation of
catalytic residues, residues thought to affect its processive nature and those thought
to be involved in target recognition, can create novel A3G enzymes. Using structure
guided selection, residues in key functional sites that are amiable to mutation will
be chosen. Individuals from the resulting libraries of mutants will be selected by
directed evolution for desired characteristics. The resulting A3G enzymes will be
examined for the relationship between their structure and function. Such engineered
A3G enzymes could be targeted to catalyse the reversion of deleterious genetic mutations.
Furthermore, engineered A3G enzymes could be used in mutational studies that call
for targeted deamination along a DNA strand, or mutational studies that call for unspecific
and high throughput DNA deamination.
PI-036
Engineering porous protein crystals as scaffolds for programmed assembly
Thaddaus Huber1, Luke Hartje1, Christopher Snow1
1Colorado State University
A key motivation for nano-biotechnology efforts is the creation of designer materials
in which the assembly acts to organize functional domains in three dimensions. Crystalline
materials are ideal from the validation perspective because X-ray diffraction can
elucidate the atomic structure. Relatively little work has focused on engineering
protein crystals as scaffolds for nanotechnology, due to the technical challenges
of coaxing typical proteins into crystallizing, and the likelihood of disrupting the
crystallization process if changes are made to the monomers. We have circumvented
these limitations by installing guest protein domains within engineered porous crystals
(∼13 nm pore diameter) that have been rendered robust using covalent crosslinks. The
retention of the scaffold structure despite changes to the solution conditions and
macromolecule uptake can be validated through X-ray diffraction.
We have engineered scaffold crystals for the non-covalent and covalent capture of
guest macromolecules. By controlling the reversible loading and release, we can prepare
“integrated” crystals with spatially segregated guest loading patterns. As assessed
using confocal microscopy, such host-guest crystals are highly stable. Ultimately,
the resulting crystals may serve as a robust alternative to DNA assemblies for the
programmed placement of macromolecules within materials.
PI-037
Engineering ultrasensitive protein probes of voltage dynamics for imaging neural activity
in vivo
Francois St-Pierre1,2, Michael Pan1,2, Helen Yang3, Xiaozhe Ding1,2, Ying Yang1,2,
Thomas Clandinin3, Michael Lin1,2
1Department of Bioengineering, Stanford University, 2Department of Pediatrics, Stanford
University, 3Department of Neurobiology, Stanford University
Nervous systems encode information as spatiotemporal patterns of membrane voltage
transients, so accurate measurement of electrical activity has been of long-standing
interest. Recent engineering efforts have improved our ability to monitor membrane
voltage dynamics using genetically encoded voltage indicators. In comparison with
electrophysiological approaches, such protein-based indicators can monitor many genetically
defined neurons simultaneously; they can also more easily measure voltage changes
from subcellular compartments such as axons and dendrites. Compared with genetically
encoded calcium indicators, voltage sensors enable a more direct, accurate, and rapid
readout of membrane potential changes. However, several challenges remain for in vivo
voltage imaging with genetically encoded indicators. In particular, current voltage
sensors are characterized by insufficient sensitivity, kinetics, and/or brightness
to be true optical replacements for electrodes in vivo. As a first step towards addressing
these challenges, we sought to develop new voltage indicators that further improve
upon the performance of the fast voltage sensor Accelerated Sensor of Action Potentials
1 (ASAP1). In ASAP1, voltage-induced conformational changes in a natural voltage-sensing
domain perturb the fluorescence emission of a covalently linked green fluorescent
protein (GFP). Using a structure-based approach to guide mutagenesis, we discovered
several amino acids that tune the kinetics and voltage sensitivity of ASAP1. These
residues are not only located in the voltage-sensing domain, but also in the fluorescent
protein and in the linkers bridging sensing domain and GFP. Our most improved variant,
ASAP2, exhibits improved sensitivity to voltage transients such as neuronal action
potentials and subthreshold depolarizations. We sought to characterize the ability
of these new voltage sensors to monitor neural activity in vivo using laser-scanning
two-photon microscopy, a technique that allows imaging with lower autofluorescence
and deeper tissue penetration. We report that ASAP sensors were able report stimulus-evoked
voltage responses in axonal termini of the fly visual interneuron L2. ASAP sensors
enabled voltage imaging with dramatically improved temporal resolution compared to
three recently reported calcium and voltage sensors. Overall, our study reports novel
voltage indicators with improved performance and highlights how specific amino acids
can tune the performance of a protein-based fluorescent sensor. We anticipate that
these results will pave the way for further engineering of voltage sensing proteins,
and that our new sensor ASAP2 will facilitate current and future efforts to understand
how neural circuits represent and transform information.
PI-038
Assembly of armadillo repeat proteins from complementary fragments
Erich Michel1, Randall Watson1, Martin Christen1, Fabian Bumback3, Andreas Plückthun2,
Oliver Zerbe1
1Department of Chemistry, University of Zurich, 2Department of Biochemistry, University
of Zurich, 3University of Melbourne
Repeat proteins are built of modules, each of which constitutes a structural motif.
In Armadillo repeat proteins each module comprises 40 residues and contains three
helices arranged in a triangular fashion. These modules pack against each other, resulting
in an elongated shape of the protein. We recently demonstrated that complementary
fragments of a designed consensus Armadillo repeat protein (ArmRP) recognize each
other [1]. The two fragments YM2: MA, in which Y, M and A denote the N-cap, internal
repeats and the C-cap, respectively, form a 1:1 complex with a nanomolar dissociation
constant, which is essentially identical to the crystal structure of the continuous
YM3A protein. We further demonstrate that structurally intact Armadillo repeat protein
complexes can be reconstituted from fragments obtained at various split sites – essentially
after every repeat but also within repeats. The fragments display variable affinities
towards each other, depending on the split site. The low affinity of some complementary
pairs can be dramatically increased upon addition of peptide ligands. While a number
of proteins are known that can be reconstituted from fragments we believe that the
fact that Armadillo repeat proteins can be reconstituted from various complementary
fragments is novel and opens new interesting perspectives and applications in biochemistry.
Reference:
[1] R. Watson, M. Christen, F. Bumback, C. Ewald, C. Reichen, M. Mihajlovic, E. Schmidt,
P. Güntert, A. Caflisch, A. Plückthun, O. Zerbe (2014): Spontaneous Self Assembly
of Engineered Armadillo Repeat Protein Fragments into a Folded Structure, Structure,
22, 985-995.
PI-039
Engineering light-controllable kinases and Cas9 endonuclease with photodissociable
dimeric fluorescent protein domains
Xin Zhou1, Linlin Fan2, Michael Lin1,3,4
1Department of Bioengineering, Stanford University, 2Department of Chemical Biology,
Harvard University, 3Department of Pediatrics, Stanford University, 4Department of
Chemical and System Biology, Stanford University
A reliable method for generating optically controllable proteins would enable researchers
to interrogate protein functions with high spatiotemporal specificity. We recently
engineered a tetrameric fluorescent protein, Dronpa145N, that undergoes light-induced
monomerization, then developed a general architecture for light-inducible proteins
based on this light-induced transition. We created proteins whose active sites were
blocked by fused Dronpa145N domains in the dark, but would become unblocked by light.
Here we present further two extensions to this concept that together enabled the generalization
of this method to additional classes of proteins. First, we engineered a photodissociable
dimeric Dronpa (pdDronpa) with tunable affinity, faster photoswitching speed, and
decreased level of protein aggregation, enabling better performance of fusion proteins.
Second, we introduce the concept of caging a protein active site by insertion of Dronpa
domains into loops rather than strictly at the protein termini. We use the pdDronpa
system to impose optical control on kinases and the Cas9 endonuclease. The resulting
light-inducible MEK1 kinase, Raf1 kinase, and Cas9 endonuclease showed high caging
efficiency of protein activities in the dark, and robust protein activation upon light
illumination. We believe that our efforts on further improving and generalizing this
method would bring the power and benefits of light control to a broad community of
biologists.
PI-040
Exploring the evolution of folds and its application for the design of functional
hybrid proteins
Saacnicteh Toledo Patiño1, Birte Höcker1
1Max Planck Institute for Developmental Biology
The structural diversity of proteins may appear endless, nevertheless even large protein
complexes can be decomposed into protein domains and smaller sub-domain sized fragments.
Only recently, we could identify such fragments employing sequence-based comparisons
of different folds, as the TIM-barrel and the flavodoxin-like fold (Farias-Rico et
al., 2014). As an extension of this work, we compared all α/β proteins and identified
several fragments shared by different folds illustrating how nature may have achieved
structural and functional diversity from a reduced set of building blocks. Inspired
by this combinatorial concept, we searched for homologous fragments bearing active
sites to engineer a functional fold-chimera. We extracted the vitamin-B12 binding
part from methylmalonyl CoA mutase, which belongs to the flavodoxin-like fold (FL)
and used it to replace the corresponding fragment in uroporphyrinogen III synthase,
which belongs to the hemD-like fold (HDL). The new hybrid resulted in a stable and
well-folded protein whose structure was determined by X-ray crystallography. Moreover,
cobalamin-binding function was successfully transferred to the new protein from the
FL parent, which shows the advantage of using this approach for the design of new
functional proteins. In addition, profile alignments revealed sequence and structural
evidence that suggested an evolutionary path for HDL from FL by gene duplication.
To test this hypothesis, we expressed a modified C-terminal half of uroporphyrinogen
III synthase and solved its structure by NMR spectroscopy, thereby confirming the
predicted FL architecture. Altogether, our approach facilitates the detection of common
ancestry among different folds contributing to our understanding of protein development.
Furthermore, our results show how new complex proteins can be designed using fragments
of existing proteins that serve as building blocks in a Lego-like manner. We believe
that combining fragments containing existing properties will provide a successful
method for the design of novel functionalities in the future.
References:
Farias-Rico JA, Schmidt S, Höcker B (2014) Evolutionary relationship of two ancient
protein superfolds. Nature Chem Biol 10:710-5.
PI-041
Semisynthesis and initial characterization of sortase A mutants containing selenocysteine
and homocysteine
Lena Schmohl1, Felix Roman Wagner1, Michael Schümann2, Eberhard Krause2, Dirk Schwarzer1
1Interfaculty Institute of Biochemistry, University of Tuebingen, 2Laboratory of Mass
Spectrometry, Leibniz-Institut Für Molekulare Pharmakologie
Sortase A is a bacterial transpeptidase that recognizes and cleaves the sorting motif
LPxTG (where x can be any amino acid) at the threonine residue. The intermediate is
bond to active site Cys184 as thioester and subsequently ligated to an N-terminal
glycine residue of a second peptide [2]. The active site cysteine plays a key role
in the reaction mechanism and we investigated this residue in more detail by exchanging
this moiety with selenocysteine (Sec) and homocysteine (Hcy). The sortase mutants
were generated by semisynthesis using expressed protein ligation (EPL). The resulting
Cys-, Sec- and Hcy-sortase enzymes were characterized and showed a moderate 2–3-fold
reduction of activity for Sec-sortase. The activity of Hcy-sortase was barely detectable
with less than 1% of wildtype activity. The alkylation efficiency of the active site
nucleophiles correlated with the expected pKa values of Sec, Cys and Hcy. Analysis
of the pH dependency of the transpeptidation reactions showed that the activity optimum
of Sec-sortase was shifted towards more acidic conditions. These investigations provide
further insights into the reaction mechanism of sortase A and the semisynthetic enzymes
may provide new tool for further biochemical studies.
[1] Schmohl L., Wagner F. R., Schümann M., Krause E., Schwarzer D., Bioorg. Med Chem.
2015, 15, 2883-9.
[2] Frankel B. A., Kruger R. G., Robinson D. E., Kelleher N. L., McCafferty D. G.,
Biochemistry, 2005, 44, 11188-200.
PI-042
Directed evolution on FucO – structural explanations for changes in substrate scope
Käthe M. Dahlström1, Cecilia Blikstad2, Mikael Widersten2, Tiina A. Salminen1,
1Structural Bioinformatics Laboratory, Biochemistry, Åbo Akademi University, 2Department
of Chemistry, Uppsala University
Propanediol oxidoreductase from Escherichia coli (FucO) uses NADH/NAD+ as cofactors
to catalyze the conversion of S-lactaldehyde to S-1,2-propanediol and vice versa.
FucO is an attractive enzyme in the search for possible biocatalysts producing α-hydroxy
aldehydes, which are important for the synthesis of natural products and synthetic
drugs. Enzymes catalyzing these types of reactions are unique in catalytic power and
stereoselectivity. The usage of FucO in synthetic industry is limited by the restricted
substrate scope, which makes FucO inactive with larger phenyl-substituted alcohols.
We used re-engineering and directed evolution to enable FucO to catalyze the regio-
and enantioselective oxidation of arylsubstituted vicinal diols, such as phenylpropanediols,
into α-hydroxy aldehyde products. We mutated amino acids considered to restrict the
entry into the active site, and modeled the mutants that were most active with the
substrates phenylacetaldehyde and S-3-phenyl-1,2-propanediol and performed docking
studies with them. As expected, our experimental and in silico results show that the
mutations enlarge the active site cavity and enable the mutant enzymes to accommodate
the new substrates. We also found specific amino acids in the active site, which need
to be conserved to allow the substrates to make stabilizing interactions. Interestingly,
an asparagine residue makes the mutant enzymes able to discriminate between phenylacetaldehyde
and S-3-phenyl-1,2-propanediol. In conclusion, we successfully re-engineered the specialist
enzyme FucO to accept also bulkier molecules as substrates, thereby making it more
useful for industrial purposes.
PI-043
Aided-Crystallization of the artificial protein Octarellin V.1 by alpha-Reps and nanobodies
Maximiliano Figueroa1, Mike Sleutel2, André Matagne3, Christian Damblon4, Els Pardon2,
Marielle Valerio-Lepiniec5, Philippe Minard5, Joseph Martial1, Cécile Van de Weerdt1
1GIGA-Research, University of Liège, 2Structural Biology Brussels, Vrije Universiteit
Brussel, 3Laboratoire d’Enzymologie et Repliement des Protéines, University of Liège,
4Department of Chemistry, University of Liège, 5Institute for Integrative Biology
of the Cell, Université de Paris Sud
One way to gain insight into the sequence-structure-function relationship in proteins
is to de novo design artificial proteins. Despite impressive successes in de novo
protein design, designing a folded protein of more than 100 amino acids still remains
a challenge. Using this approach, an idealized (beta/alpha)8 fold protein was designed
leading to the production of a protein of 216 amino acids (Octarellin V). This protein
showed a low solubility and stability. Through directed evolution we produced a soluble
variant, Octarellin V.1. The biophysical characterization of Octarellin V.1 shows
a well folded monomeric and thermostable protein with a Tm over 90°C. However, after
several screenings, we could not find crystallization conditions for this protein.
As an alternative, we decided to co-crystallize Octarellin V.1 with a protein partner
that helps the crystallization process. We used 2 protein partners: alpha-Reps and
nanobodies. The first one is characterized to interact through a large surface contact,
whereas the second is characterized to recognize an specific small epitope. Crystallization
of both complexes was performed successfully by vapor diffusion and the structures
were solved. The experimental structures correspond to the first for an artificial
protein of this size and it will allow to criticize the computational design of the
Octarellin V.
PI-044
Generation of synthetic antibodies against membrane proteins in nanodiscs for use
in structural biology
Pawel K. Dominik1, Marta T. Borowska1, Olivier Dalmas1,2, Sangwoo S. Kim1, Dawid Deneka1,
Eduardo Perozo1, Robert J. Keenan1, Anthony A. Kossiakoff1
1Department of Biochemistry and Molecular Biology, The University of Chicago, 2Department
of Structural Biology
Establishing the link between structure, dynamics, and function of membrane proteins
remains elusive. Previous efforts have presented a number of technical challenges
because the relevant functional states of membrane proteins are transient, making
it difficult to study them using high-resolution biophysical methods. Here, we describe
a robust strategy for generating a class of high performance antibody-based affinity
reagents that have proven useful in determining the structures of relevant functional
states of membrane proteins. These reagents are Fab fragments that are generated by
phage display from fully synthetic libraries and are called synthetic antibody fragments,
or sABs. We have developed phage display sorting strategies that can trap a desired
conformational state, making it accessible to structural analysis, or target a particular
epitope on the protein surface. However, to maximize this technology for membrane
proteins, several limitations of phage display sorting in detergent formats had to
be overcome, the greatest being that using detergents can produce non-native conformational
biases. We sought to address these limitations by embedding membrane proteins into
nanodiscs, soluble lipid-filled discoidal particles, to better mimic the native membrane
environment. Nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing
stimuli such as ligands, ions and pH during phage display selections. We have established
and validated an improved protocol using two membrane protein systems: 1) Mj0480,
an archaeal membrane protein of unknown function, and 2) CorA, a pentameric magnesium
ion channel. Using Mj0480, we compared the nanodisc protocol with the standard method
performed in detergent, and as an important byproduct, we characterized the influence
of the membrane protein environment on the apparent affinity of sABs to their cognate
antigen. Using CorA, we developed a more sophisticated sorting strategy resulting
in a variety of sABs specific to either the open or closed conformation of the channel.
Finally, using sABs as crystallization chaperones we obtained the structure of Mj0480
at 3.5Å resolution, and crystallized CorA in several new conditions.
PI-045
A novel drug delivery system for poorly water-soluble anti-tumor drug SN-38 utilizing
intravital transporter protein
Masatoshi Nakatsuji1, Haruka Inoue1, Masaki Kohno1, Mayu Saito1, Syogo Tsuge1, Shota
Shimizu1, Osamu Ishibashi1, Takashi Inui1
1Graduate School of Life and Environmental Sciences, Osaka Prefecture University
Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily,
and binds a large variety of small hydrophobic molecules. Using this function of L-PGDS,
we have already reported the feasibility of L-PGDS as a novel drug delivery vehicle
for the poorly water-soluble drugs [1]. SN-38, 7-ethyl-10-hydroxy-camptothecin, is
a semi-synthetic analogue of anti-cancer alkaloid camptothecin that targets DNA topoisomerase
I. Despite of the potent anti-tumor activity, however, SN-38 was not used directly
in a clinical practice due to its poor water solubility. Thus, irinotecan hydrochloride
(CPT-11), which is the water-soluble prodrug of SN-38, is used for the cancer treatment.
However, CPT-11 shows approximately 0.1% cytotoxic activity of SN-38 against the various
cancer cell lines in vitro, and its metabolic conversion rate is 10% of the original
volume of CPT-11. Here, we show the development of the drug delivery system utilizing
L-PGDS, which enables a direct clinical usage of SN-38. First, we investigated the
effect of L-PGDS on the solubility of SN-38. In the presence of 2 mM L-PGDS, the concentration
of SN-38 was 1.7 mM, which was 1,130-fold as compared with that in PBS. Then, we carried
out isothermal titration calorimetry measurements to investigate the detailed binding
mode of SN-38 to L-PGDS. As a result, it was revealed that L-PGDS binds three molecules
of SN-38, and the dissociation constant value was 60 ± 4.0 µM. In vitro growth inhibition
assay revealed that SN-38/L-PGDS complex showed the high anti-tumor activity against
three human cancer cell lines, Colo201, MDA-MB231, and PC-3. The calculated IC50 values
of SN-38/L-PGDS complex on the cell growth of Colo201, MDA-MB231, and PC-3 cells were
35 ± 6.5, 900 ± 190, and 10 ± 1.5 nM, respectively. In addition, the anti-tumor activity
of SN-38 after the administration of SN-38/L-PGDS-complex was evaluated in the Colo201
human colorectal tumor xenograft model. The intravenous administration of SN-38/L-PGDS
complex at doses of 1.0, 2.0 or 2.8 mg/kg/d every other day for 2 weeks showed a pronounced
anti-tumor activity, while the administration of CPT-11 at a dose of 4.0 mg/kg/d did
not show any anti-tumor activity. Finally, in order to estimate the side effects of
SN-38/L-PGDS complex, we performed histopathological analysis in the small intestine.
The intestinal mucosa of mice administered with SN-38/L-PGDS complex at a dose of
2.8 mg/kg/d using the same administration schedule in the growth inhibition assay
showed the preservation of the villi and the crypt architecture, which was similar
to that of PBS administered group. These results indicated that SN-38/L-PGDS complex
did not induce the intestinal lesion. In conclusion, L-PGDS could improve the solubility
of SN-38, and the intravenous administration of SN-38/L-PGDS complex showed a potent
anti-tumor activity on xenograft tumor model. Thus, we believe that L-PGDS is a novel
and valid drug delivery vehicle for SN-38.
[1] Fukuhara, A. et al. (2012) J. Control. Release 158, 143-150
PI-046
Intrinsic Disorder as Biomimetic Strategies for the Introduction of Hill-Type Cooperativity
into Biomolecular Receptors
Anna Simon1, Alexis Vallée-Bélisle2, Francesco Ricci3, Kevin Plaxco1,4,5
1Biomolecular Science and Engineering Program, UC Santa Barbara, 2Département de Chimie,
Université de Montréal, 3Dipartimento di Scienze e Tecnologie Chimiche, University
of Rome, Tor Vergata, 4Department of Biochemistry and Chemistry, 5Center for Bioengineering
Control over the sensitivity with which artificial biomolecular receptors respond
to small changes in the concentration of their target ligand is critical for the proper
function of many cellular processes. Such control could likewise be highly useful
in artificial biotechnologies in which highly responsive behavior is of value, such
as biosensors, genetic logic gates, and “smart” materials and delivery devices. In
nature, the control of molecular responsiveness is often achieved using “Hill-type”
cooperativity, a mechanism in which sequential binding events on a multivalent receptor
are coupled such that the first enhances the affinity of the next, producing a steep,
higher-order dependence on target concentration. Here we use an intrinsic-disorder-based
mechanism that can be implemented without requiring detailed structural knowledge
to rationally introduce this potentially useful property into several normally non-cooperative
biomolecules. To do so we fabricate a tandem repeat of the receptor that is destabilized
(unfolded) via the introduction of a long, unstructured loop. The loop spatially separates
the two sets of the two halves of the binding sites, preventing a complete binding
site that enables target molecule binding without prior closure of the loop. Thus,
the first binding event requires the energetically unfavorable closing of this loop,
reducing its affinity relative to that of the second binding event, which, in contrast
occurs at a pre-formed site. Using this approach we have rationally introduced cooperativity
into three unrelated aptamers, achieving in the best of these a Hill coefficient experimentally
indistinguishable from the theoretically expected maximum. The extent of cooperativity,
and thus the steepness of the binding transition, are, moreover, well modeled as simple
functions of the energetic cost of binding-induced folding, speaking to the quantitative
nature of this design strategy.
PI-047
Essential and non-essential amino acid species for an ancestral protein
Satoshi Akanuma1
1Faculty of Human Sciences, Waseda University
The translation system is an essential element for life because it links genetic information
embedded in genes to functional molecules, proteins. The modern genetic code, which
encodes the standard 20 amino acids (and three terminations) using 64 triplet codons,
is shared by most of the extant organisms on the earth. A number of theories have
been proposed for the origin and evolution of the genetic code, and these theories
suggest that only a fewer amino acids were used in primitive proteins and later the
amino acid repertoire gradually increased up to 20 through the course of evolution.
If so, one would wonder how many number of and which types of amino acids were involved
in the primitive proteins. I have begun to address this issue experimentally. I first
resurrected several ancestral proteins and then restricted the amino acid usage of
one of the resurrected proteins. I targeted nucleoside diphosphate kinase (NDK) that
catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside
diphosphate. NDK may have arisen early because at least one gene that encodes NDK
is present in most extant organisms. The first step in the reconstruction of ancestral
NDK sequences is to prepare multiple amino acid sequence alignments using homologous
sequences of NDK from extant species. Then, phylogenetic trees were built. Ancestral
sequences of NDK that represent the last common ancestors of Archaea and of Bacteria
were reconstructed using the information contained in the predictive phylogenetic
trees. The reconstructed ancestral kinases are extremely thermally stable [Akanuma
et al., 2013]. Then, using the most thermally stable ancestral NDK, Arc1, as the starting
molecule, I restricted its amino acid usage. Arc1 does not contain any cysteine residue
and therefore consists of 19 amino acid species. I completely replaced one of the
19 amino acid species by other amino acid species and thus created 19 proteins each
of which consisted of 18 amino acid species. Then, I evaluated the stabilities and
activities of the resulting 19 Arc1 variants to assess the individual contributions
of the 19 amino acid species. As the result, I found that the 19 amino acid species
do not equally contribute to the stability and activity of Arc1 and that some amino
acid species can be easily lacked but others are important or essential for its stability
and function. The result clearly shows that the full amino acid species are not necessarily
essential and supports the hypothesis that proteins in the early stage of evolution
were made from a reduced amino acid set.
Akanuma et al., Proc. Natl. Acad. Sci. USA 110, 11067–11072 (2013)
PI-048
De novo design of protein-protein interaction using hydrophobic and electrostatic
interactions
Sota Yagi1, Satoshi Akanuma2, Akihiko Yamagishi1
1Tokyo University of Pharmacy and Life Sciences, Department of Applied Life Scien,
2Waseda University, Faculty of Human Sciences
The protein surface recognition for protein-protein interactions (PPI) is involved
in signal transduction, immune reaction, and creation of the nanostructures in living
cells. The methods for rational designing of PPI that could provide non-antibody scaffolds
and nanostructured materials are required for the therapeutic and nanotechnological
applications. Although there have been some successful rational designs with computational
methods, it is still difficult to design freely the PPI onto arbitrary proteins. The
reason for this limitation is decreased solubility in the designed protein due to
the additional hydrophobic residues in order to drive PPI. Another reason is a limited
set of design modes by which proteins can interact, because the target proteins have
individual surface structures. Therefore, many methods of constructing an interface
for numerous target scaffold proteins without loss of their solubility are necessary.
Surface exposed α-helices are often observed in natural globular proteins. Moreover,
there are many examples for naturally occurring oligomeric proteins where an α-helix
from each subunit interacts to form an intermolecule coiled coil. Further, the works
related to designing of artificial helical bundle reported by the several other groups
have provided information about how to generate and tune the interaction between α-helices.
Therefore, a surface exposed α-helix would be a good target for designing a de novo
interface onto the scaffold protein. Here we engineered two different proteins, sulerythrin
and cys-LARFH, to form the cys-LARFH–sulerythrin dimer–cys-LARFH heterotetramer via
an intermolecular helix-helix interaction. Wild-type sulerythrin forms a dimeric eight-helix
bundle. Cys-LARFH is a designed monomeric protein that forms four-helix bundle containing
interhelical S-S bonds. Both sulerythrin and cys-LARFH are extremely thermostable.
To design protein-protein interfaces onto the individual proteins, we first introduced
six leucines to the two α-helices of sulerythrin and three leucines to a α-helix of
cys-LARFH. As expected, the introduction of the hydrophobic amino acids reduced their
solubilities. To recover the solubility, we then introduced six aspartates or glutamates
around the hydrophobic surface of the sulerythrin (hereafter referred to as 6L6D or
6L6E). Similarly, three arginines were introduced around the artificial hydrophobic
surface of the cys-LARFH (hereafter referred as IV-3L3R). The solubilities of the
mutants with the hydrophobic interface and additional charged residues were recovered
their solubility. In addition, the sulerythrin mutants 6L6D and 6L6E exist mainly
as dimer. The cys-LARFH mutants IV-3L3R, also exists as monomer. We then examined
the interaction between 6L6E or 6L6D and IV-3L3R. A pull-down experiment, in which
Co2+ beads bound to either His-tagged cys-LARFH and IV-3L3R were used to pull down
wild-type sulerythrin, 6L6D, or 6L6E, demonstrates that 6L6D or 6L6E specifically
interacts to IV-3L3R. Furthermore, when analysed by size exclusion chromatography,
the dominant peaks of the mixture of 6L6D and IV-3L3R appeared at the volume expected
for the heterotetrameric complex. Thus we successfully created the de novo PPI by
using a very simple concept involving hydrophobic interaction in combination with
charge interactions.
PI-049
In vitro selection of liposome anchoring peptide by cDNA display
Naoto Nemoto1, Ryoya Okawa1, Yuki Yoshikawa1, Toshiki Miyajima1, Shota Kobayashi1
1Graduate School of Science and Engineering, Saitama University
A liposome-anchoring peptide (LA peptide) was selected against liposomes composed
of dioleoyl-sn-glycero-3-phosphocholine (DOPC) by in vitro selection using cDNA display
method. The selected peptide LA peptide consists of the N-terminal region (hydrophobic)
and the C-terminal region (basic) in a characteristic manner. Thus, LA peptide was
synthesized chemically and the interactions between LA peptide and particular types
of liposomes were investigated and confirmed by confocal laser scanning microscopy.
PI-050
Designing of a novel platinum-binding amino acid sequence on a protein surface
Asumi Kaji1, Hiroya Niiro1, Satoshi Akanuma2, Tetsuya Uchida1, Akihiko Yamagishi1
1Tokyo University of Pharmacy and Life Sciences, 2Waseda University
Designing of a novel interaction between a metal and a protein is a key to create
hybrid materials between organic and inorganic materials. For example, in a glucose
biosensor, which is widely used for measuring glucose concentration in blood, glucose
oxidoreductase molecules are immobilized on a platinum electrode by polyacrylamide
gel. A metal-binding tags that is added to the N- or C-terminus of a protein is also
used for fix the protein to a metal. However, a technique to create a metal binding
site on a desired position of a protein has not been invent. If such a technique would
be established, the technique would contribute to developing and improving biosensors
and to producing new bionanoelectronic materials. In this study, we created a platinum-binding
site on a loop located at a protein surface. We used an artificial protein, LARFH,
that had been synthesized by connecting four identical alpha helices originated from
the C-terminal segment of the Escherichia coli Lac repressor with three identical
loops. We randomized the Ser, Gly, Gln, Gly, Gly, Ser sequence within one of the inter-helical
loops and then selected for binding to platinum by a T7 phage display system. Most
of the selected LARFH variants contained the Tyr, Lys, Arg, Gly, Tyr, Lys (YKRGYK)
sequence in the randomized segment. We then evaluated the affinity of the LARFH variant
to platinum by means of Quartz Crystal Microbalance analysis. We found that the variant
binds to platinum more strongly than does the original LARFH. In the annual symposium,
we will also report about the affinity of the isolated YKRGYK sequence to platinum
and about the crucial role of the first tyrosine in binding to platinum.
PI-051
Engineering of an isolated p110α subunit of PI3Kα permits crystallization and provides
a platform for structure-based drug design
Alexei Brooun1, Ping Chen1, Ya-Li Deng1, Simon Bergqvist1, Matthew Falk1, Wei Liu1,
Sergei Timofeevski1
1Oncology Structural Biology, Worldwide Research and Development, Pfizer Inc Structural
Biology, Worldwide Research and Development, Pfizer Inc, 3Oncology Structural Biology,
Worldwide Research and Development, Pfizer Inc, 4Oncology Research Unit, Worldwide
Research and Development, Pfizer Inc, 5Oncology Research Unit, Worldwide Research
and Development, Pfizer Inc, 6Oncology Structural Biology, Worldwide Research and
Development, Pfizer Inc, 7Oncology Research Unit, Worldwide Research and Development,
Pfizer Inc
PI3Kα remains an attractive target for development of anticancer targeted therapy.
A number of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85
regulatory subunit have been reported, including a few small molecule co-crystal structures,
but the utilization of this crystal form is limited by low diffraction resolution
and a crystal packing artifact that partially blocks the ATP binding site. Taking
advantage of recent data on the functional characterization of the lipid binding properties
of p110α, we designed a set of novel constructs allowing production of isolated stable
p110α subunit missing the Adapter Binding Domain (ABD) and lacking or featuring a
modified C-terminal lipid binding motif. While this protein is not catalytically competent
to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated
by direct binding studies with a pan-PI3Kα inhibitor. Additionally, we determined
apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65
Å and 2.85 Å respectively. Comparison of isolated p110α (105-1048) with the p110α/p85
complex reveals a high degree of structural similarity, which validates suitability
of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal
form of p110α readily accommodates the binding of non-covalent inhibitor by means
of a fully accessible ATP site. The strategy presented here can be also applied to
structural studies of other members of PI3KIA family.
PI-052
Identification of structural determinants involved in the differential conformational
changes of EF-hand modules
Emma Liliana Arevalo Salina1, Joel Osuna Quintero1, Humberto Flores Soto1, Gloria
Saab Rincón1
1Instituto de Biotecnología, Universidad Nacional Autónoma de México
Identification of structural determinants involved in the differential conformational
changes of EF-hand modules Calcium signals are regulated by several proteins, most
of which belong to the EF-hand superfamily. The EF-hand motif is formed by a helix-loop-helix
that binds calcium through its loop1. These motifs occur in adjacent pairs, forming
a single globular domain which is the basic structural and functional Ca2+ binding
unit. The proteins in this family can be classified as calcium sensors or modulators,
according with their function. The first group undergoes a major conformational change
upon calcium binding, while the second one remains practically unchanged1,2. To explain
the biophysics behind the different behavior of these proteins upon Ca2+ binding,
we have sought to identify structural determinants that could account for these features,
especially for the difference in the conformational change. We examined the primary
structure from two EF-hand motifs: a sensor EF-hand from chicken troponin C (SCIII)
and a modulator EF-hand from bovine Calbindin D9k (ClbN). The main differences were
in the binding Ca2+ loop and a group of charged residues in the H2 helix of the modulator
EF-hand. Then, we constructed chimeric ClbN motifs containing the loop or the loop
and H2 from SCIII motif (H1ClbNSCIII and H1H2ClbNSCIII). These constructs were analyzed
using a reporter system that discriminates EF-hand-sensor motifs from signal-modulators
at the single-motif level. This reporter is based on the fusion of genes codifying
for the EF-hand and the prephenate dehydrogenase from E. coli (TyrA), a protein which
is active only as a dimer. Isolated EF-hand motifs have the ability to homo-dimerize
and in the fusion can stabilize and activate TyrA. The sensor motif exhibits a conformational
change by binding calcium and in doing so, destabilizes the dimeric conformation of
TyrA and virtually eliminates its activity. In the modulators, on the other hand,
the rather small conformational change only gives rise to a decreased TyrA activity.
Both constructed chimeric EF-hand fusions showed a loss of activity upon Ca2+ binding,
indicating that the 12 residues connector of the sensor EF-hand from SCIII is sufficient
to confer the conformational change. In addition we used CD and extrinsic fluorescence
spectroscopies to analyze any conformational change in the H1H2ClbNSCIII and H1ClbNSCIII
isolated modules, not finding any difference between the Ca2+ free and Ca2+ bound
chimeras, suggesting that the change in activity of the reporter protein is due to
a change in the orientation of the helices in the EF-hands induced by calcium. The
effect of Ca2+ binding of the chimeras in the context of the entire Calbindin D9k
protein is under investigation.
1 Nelson, M. R., Chagot, B. & Chazin, W. J. Encyclopedia of Life Sciences. JohnWiley&Sons,Ltd,
2010
2 Gifford, J. L., Walsh, M. P. & Vogel, H. J. Structures and metal-ion-binding properties
of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem. J. 405, 199-221. 2007
PI-053
Mapping side chain interactions at the N- and C-termini of protein helices
Nicholas Newell1
1Independent Researcher
Mapping side chain interactions at the N- and C-termini of protein helices Nicholas
E Newell, Independent Researcher Interactions involving one or more amino acid side
chains near the ends of protein helices stabilize helix termini and shape the geometry
of the adjacent loops, contributing to supersecondary structure. Side chain structures
that have been identified at the helical N-terminus include the Asx/ST N-caps, the
capping box, and hydrophobic and electrostatic interactions. At the C-terminus, capping
is often achieved with main-chain polar groups, (e.g. the Schellman loop), but here
also particular side chain motifs clearly favor specific loop geometries. Key questions
that remain concerning side chain interactions at helix termini include: 1) To what
extent are helix-terminal motifs that include multiple amino acids likely to represent
genuine cooperative interactions between side chains, rather than chance alignments?
2) Which particular helix-terminal loop geometries are favored by each side chain
interaction? 3) Can an exhaustive statistical scan of a large, recent dataset identify
new side chain interactions at helix termini? In this work, three analytical tools
are applied to answer the above questions for both N- and C-termini. First, a new
perturbative least-squares 3D clustering algorithm is applied to partition the helix
terminal structures in a large (25,000 example), low-redundancy PDB dataset by loop
backbone geometry. The clustering algorithm also generates a set of structural exemplars,
one for each cluster, that is used to represent the most important loop geometries
at each terminus. Next, Cascade Detection (Newell, Bioinformatics, 2011), an algorithm
that detects multi-amino acid cooperativities by identifying overrepresented sequence
motifs, is applied to each cluster separately to determine which motifs are most important
in each loop geometry. Finally, the results for each motif are displayed in a CapMap,
a 3D conformational heatmap that depicts the distribution of motif abundance and overrepresentation
across all loop geometries by projecting these quantities onto the structural exemplars
generated by clustering. The CapMap reveals the loop conformations most favored by
a motif. Actual structures from the clusters corresponding to these favored conformations
are then examined in a structure browser to characterize the side chain interaction
associated with the motif. This work identifies a ’toolkit’ of side chain motifs which
are good candidates for use in the design of synthetic helix-terminal loops with specific
desired geometries, because they are used in nature to support these geometries. Highlights
of the analysis include determinations of the favored loop geometries for the Asx/ST
motifs, capping boxes, big boxes, and other previously known and unknown hydrophobic,
electrostatic, H-bond, and pi-stacking interactions. A goal of future work is to make
these results available in a structurally-addressable database that would enable researchers
to immediately retrieve the side chain interactions most compatible with a desired
loop geometry.
PI-054
Generation of fluorescent protein-tagged gp120 mutants to analyze the intracellular
distribution of HIV-1 envelope protein
Shuhei Nakane1, Zene Matsuda3
1Green Earth Research Center, Green Earth Institute Co., Ltd., 2Res Ctr for Asian
Infect Dis, Inst of Med Sci, the Univ of Tokyo, 3Lab of Struct Virol and Immunol,
Institute of Biophysics, CAS
HIV-1 is a causative enveloped virus of AIDS. Its envelope protein (Env) has two non-covalently
associated subunits, gp120 and gp41, which are proteolytically processed from a gp160
precursor. The gp120 subunit is a surface protein and gp41 is a transmembrane protein.
The gp120 and gp41 subunits are responsible for the receptor recognition and membrane
fusion, respectively. The cytoplasmic tail (CT) of gp41 is about 150 amino acids long
and is believed to play a critical role in intracellular trafficking of Env. To visualize
dynamic trafficking, the C-terminus of gp41 has been tagged with fluorescent proteins
such as GFP. However, tagging of CT may cause a concern to affect the interactions
between the CT and cellular proteins that are involved in intracellular trafficking.
To avoid this problem, here we tried to insert GFPopt, a GFP variant, into five variable
regions of gp120. We have analyzed the phenotypes of Env mutants, such as the cell
surface expression, processing of gp160, membrane fusion activity, and virion incorporation.
Among 5 variable regions of gp120, the V3 region was most sensitive to insertion.
V1/V2 region was less sensitive than V3. Consistent with the recently revealed structure,
exteriorly located V4 and V5 were highly tolerant to insertion. We used the mutant
with the GFP insertion in the V5 region to analyze the intracellular distribution
of Env with and without CT. We found that deletion of CT increased the presence of
vesicles colocalized with late endosome markers. This is consistent with the hypothesis
that the CT region contains a motif regulating intracellular trafficking. Our results
showed that Env with GFPopt insertion in its gp120 subunit is a useful tool for the
study of intracellular dynamics of HIV-1 Env. These mutants would also be useful to
trace the fate of virus particles during infection.
PI-055
NGS-guided phage panning: Comparison to conventional panning strategy
Buyung Santoso1, Dorain Thompson1, John Nuss1, John Dwyer1
1Ferring Research Institute
Phage display is a powerful tool for generating binders to a target protein. Multiple
rounds of panning with conventional phage display strategies typically result in a
number of hits, which are then individually screened using in vitro assays. Clones
screened at this stage are a combination of specific binders, sequences that are selected
due to amplification bias, and non-specific binders. If the number of specific clones
is low relative to the non-specific sequences, a larger number of clones have to be
screened to ensure sufficient diversity of early leads. With the advent of next generation
sequencing (NGS) technology, we aim to test whether we can increase the diversity
of specific hits and decrease the number of non-specific sequences. In our experiment,
four rounds of conventional panning produced ten peptide binders to target protein.
NGS analysis after two rounds of panning was done in parallel, yielding more than
ten thousand sequences, ranked by abundance. All ten binders from conventional panning
were found in the top 150 most abundant NGS hits. More importantly, additional hits
were found in NGS analysis but not in conventional panning, highlighting this strategy
as a promising alternative for hit discovery with the significant upside of more diverse
and higher affinity leads.
PI-056
Fully Automated Mini, Midi, and Maxi plasmid prep on the autoplasmid MEA instrument
Carrie Huynh1, Lee Hoang1, Chris Suh1, Jonathan Grambow1
1PhyNexus In San Jose
Numerous processes in pharmaceutical development, including construct screening, structural
genomics, protein engineering and expression optimization among others, require the
use of higher throughput plasmid DNA purification. The majority of issues encountered
in Mini, Midi, and Maxiprep purification kits involve flocculate removal following
alkaline lysis, and there is currently no easy way to produce large amounts of plasmid
DNA without the addition of complicated and time consuming clarification steps. The
existence of a hassle-free automated system that is not restricted by sample size
would significantly help in cutting time and costs during the initial processing steps
of plasmid purification. The AutoPlasmid MEA instrument provides a fully automated
solution to traditional problems faced in plasmid purifications, allowing Mini, Midi,
and Maxiprep plasmid purifications to be performed on a single instrument. The data
presented here on plasmid yield, purity, and suitability for sequencing and transfection/transformation
illustrate a new strategy for automated plasmid preps. By eliminating traditional
clarification methods, cell culture volumes between 1 - 120 mL can be processed leading
to yields ranging from 3 – 1000 μg. This flexible system was developed in order to
satisfy a wide variety of concentration and yield requirement, while eliminating the
time consuming steps previously needed to obtain similar results. The ability to perform
fully automated Mini, Midi, and Maxi plasmid preps on one instrument allows for a
customized all-in-one purification system that is not restricted by traditional clarification
methods, eliminating manual intervention, and streamlining the purification process.
PI-057
RE3volutionary computational design of symmetric proteins that biomineralize nano-crystals
Kam Zhang1, Arnout Voet1, Hiroki Noguchi2, Christine Addy2, Jeremy Tame2
1Structural Bioinformatics Team, DSSB, CLST, RIKEN, 2Drug Design Laboratory, GSMLS,
Yokohama City University
RE3Volutionary Computational Design of Symmetric Proteins That Biomineralize Nano-Crystals
Arnout RD Voet (1), Hiroki Noguchi (2), Christine Addy (2), Kam YJ Zhang (1), Jeremy
RH Tame (2) (1) Structural Bioinformatics Team, Division of Structural and Synthetic
Biology, Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro, Yokohama, Kanagawa
230-0045, Japan (2) Drug Design Laboratory, Graduate School of Medical Life Science,
Yokohama City University, 1-7-29 Suehiro, Yokohama, Kanagawa 230-0045, Japan The modular
nature of protein architectures suggests that proteins have evolved through duplication
and fusion to give rise to modular, often symmetric forms, which later diversified
under the influence of evolutionary pressure. We have developed a computational protein
design method termed REverse Engineer Evolution (RE3Volution) to create symmetrically
self-assembling protein building blocks. We have used this method to design a perfectly
symmetric β–propeller protein called Pizza. Subsequently, we have engineered a metal
binding site into this Pizza protein. This new Pizza variant carries two nearly identical
domains per polypeptide chain, and forms a trimer with three-fold symmetry. The designed
single metal ion binding site lies on the symmetry axis, bonding the trimer together.
Two copies of the trimer associate in the presence of cadmium chloride in solution,
and high resolution X-ray crystallographic analysis reveals a nano-crystal of cadmium
chloride, sandwiched between two trimers of the protein. This nano-crystal, containing
seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the
smallest reported to date. Our results indicate the feasibility of using rationally-designed
symmetrical proteins to biomineralize nano-crystals with applications in bio-nanotechnology.
PI-058
Bacillus licheniformis Trehalose-6-phosphate Hydrolase structures suggest keys to
substrate specificity
Chwan-Deng Hsiao1, Min-Guan Lin1, Long-Liu Lin2, Yuh-Ju Sun3
1Institute of Molecular Biology, Academia Sinica, 2Department of Applied Chemistry,
National Chiayi University, 3Depaertment of Life Science, National Tsing Hua University
Trehalose-6-phosphate hydrolase (TreA) of the glycoside hydrolase family 13 (GH13)
catalyzes the hydrolysis of trehalose-6-phosphate (T6P) to yield glucose and glucose-6-phosphate.
Products of this reaction can be further metabolized by the energy-generating glycolytic
pathway. Here we present the crystal structures of Bacillus licheniformis TreA (BlTreA)
and its R201Q mutant complexed with p-nitrophenyl-α-D-glucopyranoside (R201Q/pPNG)
at 2.0 Å and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar
to other GH13 family enzymes. However, detailed structural comparisons revealed that
the catalytic groove of BlTreA contains a long loop adopting a different conformation
from those of GH13 family members. Unlike the homologous regions of Bacillus cereus
oligo-1,6-glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX-5),
the active site surface potential of BlTreA exhibits a largely positive charge, contributed
by the four basic residues His281, His282, Lys284 and Lys292. Mutations at these residues
resulted in significant decreases of BlTreA enzymatic activity. Strikingly, a 281HHLK284
motif and the Lys292 residue played critical roles in BlTreA substrate discrimination.
PI-059
Crystal structure of engineered LRRTM2 synaptic adhesion molecule and a model for
neurexin binding
Anja Paatero1, Katja Rosti1, Alexander Shkumatov2, Cecilia Brunello3, Kai Kysenius3,
Prosanta Singha1, Henri Huttunen3, Tommi Kajander1
1Institute of Biotechnology, University of Helsinki, Helsinki, Finland, 2Dept of Pharmaceutical
and Pharmacological Sciences, KU Leuven, Leuven, Belgium, 3Neuroscience Center, University
of Helsinki
Synaptic adhesion molecules are key components in the development of the brain, and
in the formation of neuronal circuits, as they are central in the assembly and maturation
of the chemical synapses. Several families of neuronal adhesion molecules have been
identified such as NCAMs, neurexins and neuroligins, and in particular recently several
leucine rich repeat protein families, e.g. Netrin G-ligands, SLITRKs and LRRTMs. The
LRRTMs form a family of four proteins. They have been implicated in excitatory glutamatergic
synapse function, and were specifically characterized as ligands for neurexins in
excitatory synapse formation and maintenance. In addition, LRRTM3 and LRRTM4 have
been found to be ligands for heparan sulphate proteoglycans. We report here the crystal
structure of a stability-engineered mouse LRRTM2, with a Tm 30°C higher than the wild
type protein, while retaining its function. We localized the neurexin binding site
to the concave surface based on protein engineering, sequence conservation and prior
information on the ligand interaction with neurexins, allowing us to propose a tentative
model for LRRTM:neurexin interaction compex. Cell culture studies and binding experiments
show that the engineered protein is functional and capable of forming synapse-like
contacts. Small angle X-ray scattering data suggests that the wild type protein forms
transient dimers, which may have importance for the function. The structural and functional
data presented here provide the first structure of an LRRTM protein, and a model for
molecular mechanism of LRRTM function in adhesion.
PI-060
Computational design of phenylalanine binder
Olga Khersonsky1, Gil Benezer1, Sarel Fleishman1
1Department of Biological Chemistry, Weizmann Institute of Science
Recently, AbDesign algorithm was developed in our lab for de novo design of antibodies
(1). It is guided by natural conformations and sequences, and exploits the modular
nature of antibodies to generate an immense space of conformations, which can be used
as scaffolds for design of stable high-affinity binders. We have used AbDesign to
design a binder of phenylalanine. ∼30,000 antibody scaffolds were obtained by splicing
H3 and L3 fragments into a template (pdb ID 2brr), and subsequent optimization of
VH and VL orientation. Phenylalanine binding site, based on native phenylalanine binders,
was introduced into the scaffolds with RosettaMatch (2), and the sequences were subsequently
optimized by Rosetta enzyme design protocol (3). ∼30 designs were experimentally tested
by yeast display for binding of biotinylated phenylalanine ligand. Several designs
were found to bind the ligand, and we plan to further characterize this affinity and
improve it using directed evolution techniques. In collaboration with the group of
Prof. Johnsonn, the resulting phenylalanine binder will be incorporated in a bio-luminescent
(LUCID) sensor for phenylalanine (4). Phenylalanine monitoring device would be of
primary importance for patients with phenylketonuria, a genetic disease with phenylalanine
metabolism problem.
1. AbDesign: An algorithm for combinatorial backbone design guided by natural conformations
and sequences. Lapidoth G.D. et al, Proteins, 2015, in publication.
2. New algorithms and an in silico benchmark for computational enzyme design. Zanghellini
A. et al, Protein Sci. 2006, 15(12):2785-94.
3. De novo enzyme design using Rosetta3. Richter F. et al, PLoS One. 2011;6(5):e19230.
4. Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring. Griss
R. et al, Nat Chem Biol. 2014;10(7):598-603.
PI-061
Dimer dynamics in a cold-active enzyme: The case of alkaline phosphatase
Bjarni Ásgeirsson1, Manuela Magnúsdóttir1, Jens Hjörleifsson1, Gaetano Invernizzi2,
Elena Papaleo2
1Department of Biochemistry, Science Institute, University of Iceland, 2University
of Milano-Bicocca
Cold-adapted enzymes are interesting because of their higher catalytic activity compared
to mesophilic and thermophilic homologues. Alkaline phosphatase (AP) from a psychrophilic
Vibrio marine bacteria (VAP) has an unusual large surface loop that extends from each
of its monomers to stabilize a homodimeric structure (1). In many cold-adapted enzymes,
the loop regions are longer compared to proteins of mesophilic organisms and our aim
was to study the functional and structural role of this loop. Three substitutions
(R336L, Y346F and F355Y) were introduced within the large surface loop as directed
by 1-microsecond molecular dynamics (MD) simulations. With the R336L mutation, two
hydrogen bonds were broken that connect the loop to residues on the adjacent subunit,
and further two hydrogen bonds broken with the adjacent Q334. As a consequence, R336L
displayed a 25% higher kcat compared with wild-type and a slight decrease in the Km
value. Overall, the catalytic efficient improved by 45%. The global heat stability
(Tm) and the active site sensitivity to heat (T50%) were reduced by 6°C and 13°C,
respectively. MD simulations showed that hydrogen bonds to Arg336 are important for
long-range communication to the active site. Certain rotamers of two important residues
in the catalytic site, Ser65 and Arg129, were favored, presumably toward states more
competent for catalysis upon the replacement of Arg336 with Leu. In the Y346F variant,
removal of one hydrogen bond between the loop and the other subunit caused a small
drop in stability parameters, whereas both kcat and Km were reduced by about half,
giving similar kinetic efficiency (kcat/Km) to that of wild-type. Finally, we changed
a residue at the root of the large loop (F355Y) such that one new intersubunit hydrogen
bond could form. This variant maintained the wild-type characteristics. In conclusion,
removing hydrogen bonds connecting the major loop of one subunit to the protein surface
of the other subunit in VAP produced higher catalytic activity and this shows functional
connections between loop mobility and the active site. Our study also demonstrates
that interactions between residues in the large disordered loop and the opposite subunit
in the dimeric VAP are determinants of its stability. Thus, we managed to show that
loosening of interface contacts between the two VAP subunits by replacement of crucial
residues provides a way to orchestrate structural and kinetic dynamics in a productive
way.
Relevant publications:
(1) Helland, R., Larsen, R.L., Ásgeirsson, B. (2009) Biochim. Biophys. Acta.1794,
297-308.
(2) Papaleo, E., Renzetti, G., Invernizzi, G., Ásgeirsson, B. (2013) Biochim. Biophys.
Acta 1830, 2970-2980.
(3) Papaleo, E., Magnúsdóttir, M., Hjörleifsson, J.G., Invernizzi, G., Ásgeirsson,
B. (2015). Modulation of activity and stability of a dimeric cold-adapted enzyme acting
on a disordered region at the monomer-monomer interface (Under review).
PI-062
A novel secondary structure element assembly protocol for the design of artificial
(βα)8-barrel proteins using ROSETTA
Cristina Elisa Martina1, Steven Combs2, Rocco Moretti2, Maximiliano Figueroa1, Cecile
Van De Weerdt1, Andre Matagne1, Jens Meiler2
1University of Liege, 2Vanderbilt University
The de novo design of artificial proteins arises as a stringent test of our understanding
of the relationship between sequence, structure, and function. Examples include the
design of a four α-helix bundle, a new protein topology called TOP7, and a series
of artificial (βα)8-barrels called Octarellins. However, de novo design has proven
difficult for larger proteins with more than 100 amino acids. Here we present two
methods to generate the backbone and to perform the de novo design of (βα)8-barrel
proteins through the use of the software ROSETTA; both have different advantages and
limitations. The first method for generating the backbone is knowledge-based, with
a first analysis of a non-redundant database of natural (βα)8-barrel proteins in order
to obtain statistical analysis on preferred secondary structure element length and
amino acidic propensities. With this information we use the ROSETTA CM software to
create more than 1000 models which are then ranked in term of ROSETTA energy. The
second method is performed with the ParametricDesign package of ROSETTA, in which
only geometrical information are requested (number of strands and helices, radius
of the β- and α-barrels, degree of inclination, orientation of the side chains, among
others). Both methods contain a step of loop refinement and multiple steps of sequence
design with the package ROSETTA Design, in order to find low scoring amino acid sequences
for each of the starting backbone conformations. Thousands of models will be generated
by both methods and then analyzed in term of sequence similarity, secondary and tertiary
structure prediction, and stability by molecular dynamics simulations. The 30 best
candidate sequences will be selected for the experimental verification. In order to
identify a putative successfully design, we added a metal binding site during the
design step. All the proteins will be expressed in E. coli. The solubility of the
designed proteins inside bacteria will be determined thanks to the fusion to green
fluorescence protein (GFP). Solubility, stability, secondary structure, and cooperativity
of folding will be assessed for each protein before determination of their three-dimensional
structure.
PI-063
Construction of protein capsule possessing drugs controlled release ability
Shota Shimizu1, Masatoshi Nakatsuji1, Keisuke Yamaguchi1, Yuya Sano1, Yuya Miyamoto1,
Takashi Inui1
1Graduate School of Life and Environmental Sciences, Osaka Prefecture University
Most compounds that exhibit anti-tumor activities are water-insoluble, thus limiting
their clinical use. Chemical modification of these compounds and the use of solubilizing
agents such as organic solvents, surfactants and pH modifiers improve their solubility.
However, chemical modification of compounds decreases their potency, and the use of
solubilizing agents causes toxicity in many cases. Thus, drug delivery systems (DDS)
for poorly water-soluble anti-tumor drugs which exploit liposomes, cyclodextrins,
and lipid nanoparticles have been studied intensely. In these DDS, the controlled
release of drugs from the delivery vehicle is one of the most important functions.
Selective release in target cells leads to adequate therapeutic efficacy with few
side effects. In our laboratory, we have already demonstrated that lipocalin-type
prostaglandin D synthase (L-PGDS), an intravital transporter protein, is a novel and
valid drug delivery vehicle for SN-38, a poorly water-soluble anti-tumor drug. In
this study, we generated L-PGDS-based protein capsules with a controlled-release function
by introducing a disulfide bond into the upper part of the drug-binding cavity of
L-PGDS. The intracellular concentration of glutathione (0.5∼10 mM) is known to be
substantially higher than the extracellular concentration (∼2 µM). Therefore, it is
expected that in the extracellular oxidative environment the disulfide bonds in the
protein capsule remain stable, avoiding premature release of the internal drugs during
circulation of blood, after reaching the target cells, the disulfide bonds are cleaved
in the intracellular redox-environment, and then the internal drugs are released.
We generated three kinds of protein capsules which have disulfide bonds in different
positions, W54C/W112C, K58C/H111C, K58C/W112C, based on tertiary structure information
of human L-PGDS (PDB ID: 3O2Y). Firstly, we performed circular dichroism (CD) measurements
to confirm the structure of each capsule. The CD spectra of three protein capsules
were similar to that of wild-type L-PGDS in the far-UV region. Therefore, the secondary
structures of three protein capsules were not changed from wild-type L-PGDS by introducing
the mutations. Quantitative analysis of the free thiol group in the protein capsule
by DTNB assay revealed that the intermolecular disulfide bond was formed by H2O2-induced
oxidation and cleaved by dithiothreitol-induced reduction. In addition, to investigate
the solubility of SN-38 in the presence of protein capsules, we mixed the protein
capsule of reduced-form with SN-38 suspension, and stirred at 37ºC for 48 hours. The
resulting concentrations of SN-38 in PBS with 1 mM W54C/W112C, K58C/H111C, and K58C/W112C
were 374 µM, 194 µM, and 349 µM, respectively. These values were approximately 200-fold
higher than without protein capsules. SDS-PAGE analysis showed that the bond formation
decreased in a time-dependent manner, and that new intermolecular disulfide bond was
not formed in the protein capsules after 48 hours’ incubation. From the above, we
succeeded in generating drug delivery vehicles possessing openable and closable lids
that are responsive in an oxidation-reduction environment.
PI-064
Formation of Cytochrome cb562 Oligomers by Domain Swapping
Takaaki Miyamoto1, Mai Kuribayashi1, Satoshi Nagao1, Yasuhito Shomura2, Yoshiki Higuchi3,4,
Shun Hirota1
1Graduate School of Materials Science, Nara Institutte of Science and Technology,
2Graduate School of Science and Engineering, Ibaraki University, 3Department of Life
Science, Graduate School of Life Science, University of Hyogo, 4RIKEN SPring-8 Center
Domain swapping has been of interest as a mechanism of protein oligomerization, where
a secondary structural region or a domain of one protein molecule is replaced with
the corresponding region or domain of another protein molecule. We have previously
shown that c-type cytochromes and myoglobin form oligomers by domain swapping.1,2
In this study, we show that a four-helix bundle protein cyt cb562, in which the heme
of cyt b562 is attached to the protein moiety by insertion of two Cys residues, forms
a domain-swapped dimer. Dimeric cyt cb562 was more stable than dimeric cyt b562 at
4°C, showing that attachment of the heme to the protein moiety stabilizes the domain-swapped
structure. Absorption and CD spectra of dimeric cyt cb562 were similar to the corresponding
spectra of the monomer, showing that the active site and secondary structures were
similar between the dimer and monomer. The redox potential of dimeric cyt cb562 was
also similar to that of its monomer. The dissociation temperature of dimeric cyt cb562
was 50°C, and its ΔH on dissociation to monomers was −13.3 kcal/mol (per dimer). According
to X-ray crystallographic analysis, dimeric cyt cb562 exhibited a domain-swapped structure,
where the two helices in the N-terminal region (helices 1 and 2) in a protomer and
the other two helices in the C-terminal region (helices 3 and 4) of the other protomer
interacted between each other. The heme coordination structure of the dimer was similar
to that of the monomer. We have previously shown that domain-swapped oligomers of
horse cyt c form through intermolecular hydrophobic interaction between the N- and
C-terminal α-helices at the early stage of folding.3 It has been suggested that helices
2 and 3 form first at the initial stage of folding in wild-type apo cyt b562.4 Therefore,
we propose that cyt cb562 forms a domain-swapped dimer when helices 2 and 3 interact
intermolecularly at the initial stage of folding, whereas the intramolecular interaction
of helices 2 and 3 results in formation of a monomer.
1. S. Hirota, et al., Proc. Natl. Acad. Sci. USA., 2010, 107, 12854;
2. S. Nagao, et al., Dalton Trans., 2012, 41, 11378;
3. P. P., Parui, et al., Biochemistry, 2012, 52, 8732; 4. H, Feng, et al., Proc. Natl.
Acad. Sci. USA., 2005, 102, 5026.
PI-065
A highly buried and conserved tryptophan residue close to the dimer interface in a
cold-adapted phosphatase is phosphorescent and important for activity
Jens Hjörleifsson1, Bjarni Ásgeirsson1
1Department of Biochemistry, Science Institute, University of Iceland
A highly buried and conserved tryptophan residue close to the dimer interface in a
cold-adapted phosphatase is phosphorescent and important for activity. Jens G. Hjörleifsson
and Bjarni Ásgeirsson. Department of Biochemistry, Science Institute, University of
Iceland, Dunhagi 3, 107 Reykjavik, Iceland. Alkaline phosphatase (AP) from Vibrio
G15-21 is a cold-adapted dimeric enzyme with one of the highest catalytic efficiency
reported for known APs. It contains five intrinsic tryptophan (Trp) residues and one
additional Trp located on the C-terminal StrepTag used for expression and purification.
In this study, we made several single Trp-substitutions to determine the role of each
of the Trp in the fluorescence emission spectrum. We also determined their solvent
exposure by acrylamide fluorescence quenching. The results indicate that Trp301, Trp460
and Trp475 are mostly responsible for the fluorescence emission. Quenching experiments
with acrylamide indicated that all the Trp residues were about equally accessible
for quenching, except Trp460 which was shown to be highly buried in the core of the
protein. Interestingly, the enzyme was found to be highly phosphorescent at 10°C,
having two phosphorescence lifetimes. The longer lifetime is due to Trp460. Trp460
is located close to the dimer interface and points towards a helix in the active site
where His277 binds an active-site zinc ion. In other APs, an aromatic amino acid is
conserved in the location occupied by the Trp460 residue. In most cases for cold-adapted
APs it is indeed a Trp. Interestingly, the mutation of the Trp460 to a phenylalanine
affected both stability and activity of the enzyme. kcat/KM was 10-fold lower than
for wild-type. Overall, this study reveals that Trp460 can be used as a phosphorescent
probe of local dynamics and could possibly also serve to study the dimer-monomer equilibrium
due to proximity to the dimer interface, an area clearly crucial for enzyme activity
and stability.
PI-066
Modulating protein-protein interaction with a molecular tether
Helen Farrants1, Oliver Hantschel1, Kai Johnsson1
1École Polytechnique Fédérale de Lausanne (EPFL)
High-affinity scaffolds for protein-protein interactions, such as monobodies and DARPins
can be engineered in vitro to bind to protein targets. We speculate that the affinity
for the target protein can be modulated by incorporating these evolved scaffolds and
a synthetic intramolecular tether into protein switches, in a protein construct of
composed of SNAP-tag, a monobody and a circular permutated dihydrofolate reductase.
The tether, attached to the construct via SNAP-tag, was composed of a linker and trimethoprim,
which interacts reversibly with the circular permutated dihydrofolate reductase. We
have investigated the affinity between the N-SH2 domain of the phosphatase Shp2 and
an evolved monobody in such a protein construct using a FRET assay. When the intramolecular
tether was bound the circular permutated dihydrofolate reductase (“closed” conformation),
there was an increase in the affinity of the construct to the target N-SH2. In the
presence of a small molecule competitor (“open” conformation) the affinity of the
monobody construct to its target was reverted to the value reported in the literature.
The intramolecular tether in these protein constructs combined with engineered scaffolds
for protein-protein interactions may be a general approach towards protein switches.
PI-067
LIL traptamers: artificial transmembrane proteins with minimal chemical diversity
Daniel DiMaio1, Erin Heim1, Ross Federman1, Lisa Petti1, Jez Marston1
1Yale Unversity School of Medicine
Because most proteins are long polymers of amino acids with twenty or more chemically-distinct
side-chains, there are an enormous number of potential protein sequences. Here, we
report the construction of biologically active proteins with minimal chemical diversity.
Transmembrane domains of proteins can specifically interact with other transmembrane
domains to modulate the folding, oligomerization, and function of transmembrane proteins.
For example, the bovine papillomavirus E5 protein is a 44-amino acid transmembrane
protein that transforms fibroblasts to tumorigenicity by binding directly to the transmembrane
domain of the platelet-derived growth factor β receptor (PDGFβR), resulting in ligand-independent
receptor activation and cell transformation. These studies showed that a free-standing
transmembrane domain could fold properly in cells and act in trans to modulate the
activity of a larger transmembrane protein target. Because of the relative chemical
simplicity of transmembrane domains and this ability to act even when not linked to
more complex soluble protein domains, we reasoned that short transmembrane proteins
could be used to define the minimal chemical diversity sufficient to construct biologically
active proteins. To accomplish this, we infected cultured mouse cells with a retroviral
library expressing 26-amino acid proteins consisting of an initiating methionine followed
by a randomized sequence of leucines and isoleucines, two hydrophobic amino acids
that differ only by the position of a single methyl group, and selected rare proteins
with transforming activity. We isolated numerous proteins consisting of diverse sequences
of leucine and isoleucine that cause morphologic transformation, escape from contact
inhibition and focus formation, and growth factor independence. Genetic and biochemical
analysis of these proteins indicate that like E5 they interact with the transmembrane
domain of the PDGFβR to specifically activate the receptor and transform cells. Mutational
analysis of individual proteins identified specific leucines and isoleucines required
for transforming activity, and insertion of a single isoleucine at a particular position
in a stretch of leucines is sufficient for activity. These proteins identify the minimal
chemical diversity required to generate a biologically active protein and have important
implications for biochemistry, protein evolution, protein engineering and synthetic
biology.
PI-068
Efficient Encapsulation of Enzymes in an Engineered Protein Cage
Yusuke Azuma1, Donald Hilvert1
1Laboratory of Organic Chemistry, ETH Zurich
Virus-like particles that are precisely loaded with functional cargo are an important
tool to study the effect of spatial confinement and create novel entities with application
in biotechnology and medicine. By genetic fusion to a positively supercharged green
fluorescent protein (GFP(+36)), an enzyme retro-aldolase (RA) was efficiently targeted
to the negatively charged lumen of an engineered protein cage, Aquifex aeolicus lumazine
synthase variant 13 (AaLS-13). The encapsulation is quantitative under mild aqueous
condition up to a mixing ratio of 45 guest enzymes per host cages. The chromophoric
tag is used for precisely quantifying the enzyme concentration, which allows detailed
characterization of the effect of encapsulation on the enzyme activity. The generality
of the encapsulation system was examined with 8 structurally different enzymes.
PI-069
Identification of disease-related antigen-specific human antibodies by a method that
combines biopanning and high throughput sequencing from patient-derived scFv antibody
library
Yuji Ito1, Yurie Enomoto1, Shuhei Umemura1, Aiko Fujiyama1, Ryoko Mieno1, Yukiko Kato1,
Dai-ichiro Kato1
1Graduate School of Science and Engineering, kagoshima University
Introduction and purpose: In the immune system, high affinity antibodies are generated
by selection of B cells activated by antigen-stimulation followed by additional optimization
through somatic hyper mutation of antibody genes. In the artificial antibody libraries,
such as phage libraries, selection of specific antibody clones from the library is
performed by in vitro selection process called biopanning and the subsequent binding
screening. However, in spite of high efficiency of enrichment in biopanning, there
is a possibility that we overlook the minor antigen-specific clones in the screening
because of the limitation of the number of clones employed for screening. In recent
years, high-throughput analysis of DNA sequences by the next-generation sequencer
(NGS) has become available not only for genomic analysis of organisms but also repertoire
analysis of antibodies. In this presentation, we report the successful isolation of
a variety of antigen specific antibodies from patients-derived antibody phage library
by a combination method of high throughput sequence analysis on NGS and biopanning.
Method: We constructed two kinds of human single chain Fv (scFv) antibody libraries
from pooled mRNA of five cancer patients and of a wheat allergy patient, respectively.
After biopanning against a cancer antigen or wheat allergy antigen “gluten”, the phagemid
vector DNA prepared from the pooled phages before or after biopanning was used for
PCR amplifications of VH genes, adding the index and adapter sequences for NGS analysis.
The high throughput sequencing was performed on Miseq (illumina) using MiSeq Reagent
Kits v3. After discarding the short sequences and low quality data, 5’-and 3’-reading
sequences were unified by a Merge program. The frequencies (%) of all VH sequences
were evaluated using a program based on Usearch 8.0 clustering software and the changes
of the frequency (%) of each sequence between before and after panning were assigned
as amplification rate. Results and discussion: VH sequences at each round of pooled
phages after biopanning against cancer antigen were analyzed on NGS. After three rounds
of biopanning, three clusters of antibody sequences were specifically enriched suggesting
these are specific binders. To check this, scFv gene were regenerated by PCR using
H-CDR3 specific primers and scFv-displaying phages reconstructed were subjected to
binding analysis. All three phages showed a clear specific binding to cancer antigen
in ELISA. Subsequently, to test the usefulness of this method, we applied it to identify
allergen-specific scFv from allergy patient-derived antibody phage library. The phylogenetic
tree analysis of VH sequences which showed the amplification rate higher than 2.5
by a single round of biopanning elucidated total eleven clusters of VH sequences.
The VH sequences in the two clusters with the highest amplification factor were selected
and the regenerated scFv-displayed phages were tested for binding analysis. The prepared
scFv-displayed phages and also scFv proteins showed a clear binding ability to allergen.
Thus, it is suggested that the analytical method of VH sequences on NGS before and
after biopanning is very useful to isolate a variety of disease related antigen-specific
novel antibodies quickly with high degree of certainty.
PI-070
Biochemical analysis of the recognition helix of Z-DNA binding proteins: Roles in
conformational specificity
Yang-Gyun Kim1, Xu Zheng1, So-Young Park1
1Department of Chemistry, Sungkyunkwan University
Conversion of right-handed B-DNA into left-handed Z-DNA is one of the dramatic structural
transitions in biological processes including gene regulation and chromatin remodeling.
Z-DNA binding motif, Zalpha (Zα), was first discovered from human ADAR1. Subsequently,
with sequence and structure similarity to the hZαADAR1, families of proteins including
viral E3L, interferon-induced protein DAI (ZBP1) and PKZ has been identified to have
Zα domain(s). Interestingly, the Zα domain of the E3L protein from vaccinia virus
(vvZαE3L) was confirmed to have the ability of Z-DNA-binding, but it does not have
the B-to-Z conversion activity. Here, we showed that the replacement of the α3-helix
of vvZαE3L (vvZαE3L-α3) with that of hZαADAR1 results in acquiring the ability to
converting B-DNA to Z-DNA. The detailed biochemical analysis of the α3-helix mutants
of vvZαE3L further suggested that the contribution of positively charged residues
in the C-terminal part of the α3-helix is crucial during the B-to-Z transition. In
addition, hydrophobic residues of the N-terminal part of the vvZαE3L-α3 also influence
on the B-to-Z conversion activity, possibly through forming a tightly-packed structure.
In conclusion, our results revealed the previously-unknown contribution of amino acid
residues existed in the α3-helix of the Zα domains to the B-to-Z conversion. Moreover,
it strongly implies that such residues may play important roles in initiating conformational
changes of DNA structure during the B-to-Z conversion event.
[This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future
Planning (No. 2015R1A2A2A01008367).]
PI-071
Photo switching of protein conformation
Frank D. Sönnichsen1, Matthias Lipfert1, Hauke Kobarg1, Anne Müller1, Thisbe K. Lindhorst1
1Otto Diels Institute for Organic Chemistry, Christian-Albrechts-University
The ability of switching the activity of proteins at will is of great interest from
an application point of view. One promising approach utilizes a protein modification
with an organic photochromic molecule. Linking two protein side chains with the photochrome
that undergoes a light induced conformational change, protein secondary and tertiary
structure can be stabilized or destabilized and thus the structure dependent activity
can be switched “on” and “off” by light irradiation. For this the photochrome must
fulfil several requirements. Foremost, it must possess two states of comparable stability
that differ significantly in their geometry. It must further be water soluble and
non-toxic, and should not experience fatigue phenomena upon multiple irradiations.
There are two classes of molecules that fulfil those requirements: azobenzenes and
spiropyrans. We are pursuing two different strategies for the design of photoswitchable
proteins. In the first approach we attach an azobenzene compounds to side chains of
the alpha-helical antifreeze protein Type I. The end to end distance of the photochromic
molecule is sterically compatible with the folded helix only in one form, photoisomerization
therefore switches the folding state between an active helical state and an inactive
unfolded form. In a second, more general approach we use the Trp-Cage domain as a
switching unit. The Trp-Cage is the smallest known folded protein (20 amino acids).
Its folding is induced by hydrophobic interactions of a tryptophan side chain in a
short helical segment. After modification with a photochromic molecule in appropriate
positions, its structure is rendered sensitive to the state of the chromophore. By
creating protein chimera of such a Trp-Cage and biologically active peptides with
helical propensity, we aim at conferring the light-dependent fold of the cage to the
attached peptide moiety.
PI-072
ADSETMEAS: Automated Determination of Salt-bridge Energy Terms and Micro Environment
from Atomic Structures using APBS method, version 1.0
Arnab Nayek1, Shyamashree Banerjee1, Parth Sarthi Sen Gupta1, Biswa pratap Sur1, Pratay
Seth1, Sunit Das1, Nathan A Baker2, Amal K Bandyopadhyay1
1Department of Biotechnology, The University of Burdwan, Burdwan, WB, India, 2Pacific
Northwest National Laboratory
Salt-bridges are electrostatic interactions between groups of opposite charges. Net
interaction energy (ΔΔGnet) of a salt-bridge is partitioned into bridge (ΔΔGbrd),
desolvation (ΔΔGdsolv) and protein (ΔΔGprot) energy-terms of which estimation of ΔΔGdsolv
and ΔΔGprot are only possible by computational means. Thus, general purpose Poisson-Boltzmann
Equation solver: “Delphi” (in commercial package of INSIGHT-II) and “APBS” (Open-source)
are popularly used to determine these energy-terms. Nevertheless, the computation-method
is highly involved one than other uses of these solvers. Moreover, protein-specific
salt-bridges, grid-points, center, hydrophobic-isosteres-mediated mutation-files of
original charge-radius file and others are to be worked out prior to the computation.
This might answer as to why only limited numbers of structure files (≤2% of crystal-structure-database)
are worked out till date. At this juncture, an efficient fully automated all-in-one-procedure
that could analyze large dataset in a single run would be useful. To the best of our
knowledge, such procedure is truly lacking in public domain. At this end, our fully
automated all-in-one procedure: ADSETMEAS (available freely at http://sourceforge.net/projects/ADSETMEAS/along
with detailed documentation) uses “APBS” method to compute component as well as net
energy-terms of salt-bridges and redirect compact output in excelformat. Further,
micro-environments of salt-bridges are also been reported based on the presence of
polar, dipolar, acidic, basic and hydrophobic side-chains in their proximity. The
procedure provides versatility to users in choosing a] model for computation of energy-terms
to-date available in the literature and b] method (default or advanced) for parametric
optimization in “APBS” calculations. It works in UNIX like environment including CYGWIN.
It processes all proteins present in the working directory with any number of salt-bridges
in them. A pre-released version of the procedure was successfully applied for energy-terms
on 220 salt-bridges from 22 halophilic proteins. Overall, our ADSETMEAS provides intricate
details on salt-bridge energetic from crystal structures and find application in the
field of computational structural biology. These and other results will be discussed
in the conference.
PI-073
Next generation analgesics – targeting ion channels with antibody-drug conjugates
(ADCs)
Anna Wojciechowska-Bason1, Clare Jones2, Chris Lloyd3
1Postdoctoral Fellow, ADPE, Medimmune, Cambridge, 2RIA, Medimmune, Cambridge, 3ADPE
Ion channels are common targets for chronic pain therapies. Small molecule analgesics
are widely used therapeutically, but due to poor specificity they often cause a wide
range of side effects. As a result, efficacy of existing treatments is very limited.
We believe that to achieve the required specificity and efficacy, a novel and innovative
approach is required that would combine the potency of the small molecule with the
selectivity of an antibody. Therefore, we propose to apply antibody-drug conjugates
(ADCs) to deliver small molecules or peptides to ion channels in order to specifically
modulate pain signalling pathways. Voltage-gated sodium channel Nav1.7 has a well
characterised role in the perception of pain. Here we present the activity of the
peptide Huwentoxin-IV (HWTX-IV) and small molecule inhibitors PTC-A, PTC-B and PTC-C
on voltage gated sodium channels Nav1.7 and Nav1.6. In novel findings, we report that
these inhibitors show little selectivity between the voltage-gated sodium channel
family members, Nav1.6 and Nav1.7, and that the IC50 values and the impact on channel
biophysics (voltage-dependence of activation and fast inactivation) of the inhibitors
are largely similar for both channel types. Therefore, the use of HWTX-IV and other
small molecule inhibitors of Nav1.7 for pain therapy could be dose-limited due to
side effects mediated by the inhibition of channel Nav1.6. In conclusion, we propose
that HWTX-IV and the investigated small molecule inhibitors could be used for the
treatment of pain as part of a Nav1.7 antibody-drug conjugate (Nav1.7-ADC), establishing
Nav1.7 specificity and minimising side effects.
PI-074
Semi-synthesis and Evaluation of Parasitic GPI-Anchored Proteins
Maria Antonietta Carillo1, Daniel Varon Silva1
1Max Planck Institute of Colloids and Interfaces, Biomolecular System department
Malaria is one of the most infectious diseases caused by Plasmodium species parasites.
The merozoite surface protein 1 (MSP1) is the most abundant protein on the surface
of the Plasmodium species merozoite stage, which plays an important role during the
erythrocytes invasion process [1]. MSP1 is synthesized as a ∼200-kDa glycosylphosphatidylinositol
(GPI) anchored protein precursor which is processed at the end of the schizogony into
four different fragments. The primary processing step produces a complex of four fragments
that are present on the merozoite surface. The secondary processing step at erythrocytes
invasion results in the detaching of the complex from the surface, except for the
C-terminal 19-kDa domain (MSP119), which remains anchored to the parasite surface
by the GPI moiety. In human malarial infections, the GPI is considered to be a toxin
that causes the expression of various host genes and induces a pro-inflammatory immune
response, making it a valuable candidate for the development of anti-malarial drugs.
In order to study the function of the GPI and evaluate the effects, MSP119 fragment
has been expressed, purified and anchored to the synthetic GPI molecule using protein
trans-splicing strategy based on the split intein method [2]. The role of the GPI
moiety will be studied through protein folding experiments and the effect of the anchored
protein will be evaluated in vitro in order to understand the function of the GPIs.
PI-075
Assessment of UCH-L3 Substrate Selectivity using Engineered Ubiquitin Fusions with
Varying Linker Lengths
Peter Suon, Mario Navarro, John Love
1San Diego State University, 2San Diego State University, 3San Diego State University
Assessment of UCH-L3 Substrate Selectivity using Engineered Ubiquitin Fusions with
Varying Linker Lengths Peter Suon, Mario Navarro, and John J. Love San Diego State
University The Ubiquitin Proteasome System (UPS) is a complex system composed of multiple
structural and functional elements that play key roles in cellular processes such
as signal transduction, cell cycle regulation, apoptosis, and protein degradation.
Proteins destined for degradation are first tagged with the protein, ubiquitin, which
is covalently attached to internal lysine residues. Once the target has be degraded
by the proteasome; the enzyme Ubiquitin Carboxy Hydrolase L3 (UCH-L3) is believed
to prepare ubiquitin for additional rounds of ubiquitination by cleaving small peptides
and chemical adducts from the ubiquitin C-terminus. Previously in our laboratory,
protein substrates of UCH-L3 were engineered and used to characterize UCH-L3 substrate
selectivity. The engineered substrates consisted of N-terminal monoubiquitinated test
variants derived from Streptococcal protein G (protein Gβ1) and Staphylococcal protein
A (SpAB). The thermal denaturation temperatures (Tm) of the fusion proteins were measured
using circular dichroism and span a range of over 60°C. More importantly, the rate
of hydrolysis for the fusion proteins is demonstrated to be directly correlated to
the Tm of the test variant fused to the C-terminus of ubiquitin. Previously, the engineered
substrates were designed to emulate natural ubiquitin fusions and thus did not contain
any ‘linker’ residues between the C-terminus of ubiquitin and the N-terminus of the
test protein. To explore the effects of linker length on UCH-L3 hydrolysis we are
engineering new UCH-L3 substrates that contain an unstructured 12 amino acid linker
between ubiquitin and the test protein. To further explore the catalytic efficiency
of UCH-L3 we will revisit diubiquitin (Ub-Ub), which is not hydrolyzed by UCH-L3,
and will make mutations in the hopes of generating a hydrolysable substrate. Using
rational design, the new variants will be engineered to destabilize the C-terminal
ubiquitin to determine if this results in hydrolysis of the new Ub-Ub construct. The
thermal stability of these new fusion protein substrates will be measured using circular
dichroism spectroscopy (CD) and UCH-L3 hydrolysis rates will be characterized using
existing assays. Our goal is to continue the use of engineered substrates to further
explore the catalytic properties of UCH-L3 activity and the potential role in protein
trafficking and degradation within living cells.
PI-076
Beta-hairpins: Molecular Accessories for Helical Peptide Expression
Melissa Lokensgard1, John Love1
1San Diego State Uniersity
We present a biophysical study of a suite of helical proteins that have been modified
to contain 12- and 17- amino acid additions on their termini that impart increased
resistance to degradation in E. coli recombinant expression systems. The B domain
of Staphylococcal Protein A (AB) and the homeobox DNA-binding domain from D. melanogaster
Engrailed (En) are small 3-helix bundles. These domains do not appreciably accumulate
in the E. coli BL21 (DE3) cytoplasm when expression in a pET vector is chemically
induced. This is likely due to host protein degradation/recycling factors that function
to efficiently degrade these two proteins. Addition of sequences encoding either of
two amino-terminal beta-hairpins to either the N- or C-terminus of AB and En results
in the accumulation of large amounts of these new chimeric proteins. Additionally,
destabilization of the AB or En sequence does not abolish the expression enhancement
effect of the beta-hairpin addition. We have investigated the biophysical origins
and effects of the beta-hairpin additions using circular dichroism (CD) spectroscopy,
and have determined that the added sequence does not significantly perturb the secondary
structure of AB or En, nor does it significantly influence the unfolding temperature
(Tm). While investigation into the origin of the accumulation effect is ongoing, we
hypothesize that the addition of the sequence is disruptive to recognition events
in the native protein degradation machinery in E. coli. Thus, this approach represents
both a biotechnological tool for expressing helical peptides recalcitrant to expression,
as well as a system well-suited to probing mechanisms of protein recycling and homeostasis.
PI-077
Development of a semisynthetic method for the cell surface presentation of proteins
Dorottya Németh1, Balázs Schäfer1, Éva Hunyadi-Gulyás2, Zsuzsanna Darula2, Csaba Tömböly1
1Biological Research Centre,Instute of Biochemisty,Laboratory of Chemical Biology,
2Biological Research Centre, Laboratory of Proteomics Research
Current Protein Society member: Jody McGiness; jmcginness@proteinsociety.org Cell
surface proteins have important biological functions including signal transduction,
cell adhesion or antigen presentation. A special class of these proteins are lipidated
proteins containing a glycosylphosphatidylinositol (GPI) glycolipid moiety at the
C-terminus. The lipid chains of the GPI anchor molecule are responsible for the membrane
association of the attached protein. A unique feature of GPI-anchored proteins is
that after isolation they can be reinserted into the membrane of recipient cells with
the retention of the biological function. Accordingly, the exogenous introduction
of fluorescent GPI-anchored protein analogues into cell membranes is a useful method
for visualizing the cellular traffic of membrane associated proteins and for engineering
cell surfaces. We have recently shown that cholesterol can be applied for anchoring
proteins to the plasma membrane of live cells without perturbing the membrane. In
order to introduce proteins containing covalent modifications that are not genetically
encoded, an enzymatic method was considered and fused with the C-terminal cholesterylation
method. The usefulness of the method is demonstrated via the preparation of multimeric
model proteins of 40 kDa monomers, that is an appropriate representation of the ligation
of domain size proteins.
PI-078
Transmembrane domain dimerization drives p75NTR partitioning to lipid rafts
Irmina García Carpio1, Marçal Vilar1
1Sociedad de Biofísica de España. SBE
p75 neurotrophin receptor (p75NTR), is best known for its role in mediating neuron
cell death during development or after injury but it also regulates cell proliferation,
axon guidance or survival. The key to understand its signaling could rely in its structure
and conformational states. It has been described that p75 forms disulfide-linked dimmers
through the Cys257 in the transmembrane domain which are essential for its NGF mediated
signaling. Previous studies have shown that p75 is present in lipid rafts, where it
interacts with intracellular adaptors to activate different signaling pathways. We
design several p75 mutants in the TM domain that impairs dimerization and study the
role of TM domain dimerization in lipid rafts recruitment. Our analysis suggests that
p75 TM domain dimerization influences lipid raft partitioning. These results could
be a key role to understand its signaling and processing
PI-079
Bioluminescent sensor proteins for therapeutic drug monitoring of the monoclonal antibody
Cetuximab
Martijn Van Rosmalen1, Remco Arts1, Brian Janssen1, Natalie Hendrikse1, Dave Wanders1,
Maarten Merkx1
1Laboratory of Chemical Biology/Institute of Complex Molecular Systems
Therapeutic Drug Monitoring (TDM) – adapting the drug dosage scheme to the individual
patient’s pharmacokinetic and pharmacodynamic characteristics – is still uncommon
for therapeutic monoclonal antibodies, despite preliminary studies showing its potential
benefits. One of the factors impairing TDM implementation is the lack of equipment
and trained personnel to regularly measure drug concentrations in patients receiving
treatment. Point-of-care diagnostic devices which could be used by patients themselves
or by their general practitioners would greatly advance the feasibility of TDM. Here
we present a biosensor for the therapeutic monoclonal antibody Cetuximab. We developed
a series of cyclic peptides that specifically recognize Cetuximab, covering a fourfold
range of affinities, and incorporated these cyclic peptide sequences into a set of
luminescent sensor proteins. The sensors translate cetuximab concentrations into a
change in emission color that can be read out using a mobile phone camera. Together,
these sensors can quantify cetuximab levels within the relevant therapeutic concentration
range and we propose that they can be used for Therapeutic Drug Monitoring applications.
PI-080
Genetically encoded biosensor for cell permeability of inhibitors of the p53-HDM2
interaction
Silvia Scarabelli1, Thomas Vorherr2, Kai Johnsson1
1Ecole Polytechnique Fédérale de Lausanne, 2Novartis Institute for BioMedical Research
The evaluation of the permeability across the cellular membrane is a key step in the
development of therapeutics, since it affects the distribution and the efficacy of
the latters. Reliable and versatile techniques for the determination of structural
permeability determinants of molecules and information about the entry kinetics are
still missing. We introduced in the past a class of semi-synthetic ratiometric sensor
proteins (Snifits) that has been shown to be suitable for the measurement of intracellular
metabolites concentrations. Here we describe a totally genetically encoded sensor
based on the Snifits modular design for the assessment of the cell permeability of
small molecules and peptides inhibitors of the protein-protein interaction between
p53 and HDM2. We show that our sensor detects the presence of HDM2-binding stapled
peptides in vitro, and, when expressed in mammalian cells, it responds to the perfusion
of the known small molecule HDM2 inhibitor Nutlin-3a. Moreover, experiments made with
an automated microscope show that the sensor is suitable for measuring and comparing
the kinetics of entry of different kinds of inhibitors in the cytosol of living cells.
In parallel, we are developing an HCAII-based sensor protein for the sensing of sulfonamides
and eventually their peptide derivatives. We show that the sensor responds to the
presence of different kinds of HCA-inhibitors in vitro and in perfusion experiments.
This second sensor would broaden the range of molecules and peptides whose permeability
can be studied with our tools beyond the family of the HDM2-binders. Our sensors overcome
the limitations of the already existing techniques for measurements of permeability
while offering a simultaneous measurement of the cell permeability and of the binding
efficiency of small molecules and peptides of interest.
PI-081
Archer: Predicting protein function using local structural features. A helpful tool
for protein redesign.
Jaume Bonet1, Javier Garcia-Garcia1, Joan Planas-Iglesias2, Narcis Fernandez-Fuentes3,
Baldo Oliva1
1Structural Bioinformatics Lab, GRIB, UPF, 2Division of Metabolic and Vascular Health,
University of Warwick, 3IBERS, Abersystwyth University
The advance of high-throughput sequencing methodologies has led to an exponential
increase of new protein sequences, a large proportion of which remain unannotated.
The gap between the number of known proteins and those with assigned function is increasing.
In light of this situation, computational methods to predict the function of proteins
have become a valid and necessary strategy. Here we present Archer, a server that
exploits ArchDB’s hierarchy of super-secondary structures to map GO and Enzyme functions
upon protein regions and, thus, infer the function of a protein. The server relies
on either the sequence or structure of the protein of interest and returns the mapping
of functional subclasses extracted from ArchDB. Moreover, it computes the functional
enrichment and significance of each subclass, combines the functional descriptors
and predicts the function of the query-protein. Combining the functional enrichment
analysis of the super-secondary structures with the structural classification of ArchDB,
users can select variants of the target sequence that swap the region of a super-secondary
structure by another that putatively fits in the same scaffold minimizing the effect
on the global tertiary structure. Only variants that modify the predicted function
are offered for selection, thus providing a rational, knowledge-based, approach for
protein design and functionalization. The Archer server is accessible at http://sbi.imim.es/archer.
PI-082
Light-induced interaction of protomers in bacterial phytochrome from Rhodopseudomonas
palustris
Taras Redchuk1, Evgeniya Omelina1, Konstantin Chernov1, Vladislav Verkhusha1,2
1Dept. of Biochemistry, Faculty of Medicine, University of Helsinki, 2Dept. of Anatomy
and Structural Biology, Albert Einstein College of Medicine
Phytochromes are natural photoreceptors known to regulate photosynthesis in plants,
fungi and bacteria. Phytochromes found in bacteria share common architecture and consist
of a PAS-GAF-PHY photosensory core and a C-terminal output module, responsible for
biological function. A bacterial phytochrome, BphP1, from Rhodopseudomonas palustris
undergoes reversible conversion from the far-red absorbing state (Pfr) to the red-absorbing
state (Pr) followed by the conformational change upon 740 nm light irradiation. As
most of bacterial phytochromes, BphP1 forms a dimer. It was shown that 740 nm light
causes a protomer swapping between the BphP1 dimers; and likely, the output module
is involved in this process. However, the mechanism of the light-induced swapping
is poorly studied. We tested an ability of the protomer swapping between BphP1 dimers
using pull-down biochemical assay. For this, strep-tagged BphP1 was immobilized on
Strep-Tactin sepharose beads in the presence of untagged BphP1 fused to mRuby2 at
different concentrations. After incubation, the proteins were eluted and visualized
in SDS-gel using a zinc-induced fluorescence assay. An amount of the bound to beads
protein was estimated by densitometry. It was found that more than 75% of heterodimers
(strep-tagged-BphP1 and BphP1-mRuby2) form within 2.5 h of incubation under 740 nm
light at 8-fold excess of one of the interacting partners. In darkness, the swapping
was much slower. In the similar setup we checked the amount of heterodimers after
15, 30 and 120 min of incubation. No difference was observed for different time points,
suggesting that the protomer swapping is relatively fast process. Next, a role of
the C-terminal effector domain of BphP1 in the light-induced interaction was studied.
For this, kinetics of the Pfr-to-Pr transition was analysed by measuring of absorbance
at 680 nm and 740 nm for full-length BphP1 and a BphP1 mutant with the deleted C-terminal
domain. While full-length BphP1 showed the normal Pfr-to-Pr transition, absorbance
of the mutated BphP1 at 680 nm did not raise. However, 740 nm absorbance changes were
similar for both proteins; and surprisingly, the similar dark relaxation kinetics
was observed. We propose that the impaired Pfr-to-Pr transition is caused by restricted
Pr conformation in the mutant rather than by fast Pr-to-Pfr relaxation. Understanding
the mechanisms of the BphP1 light-induced structural changes and the protomer interaction
should advance engineering of bacterial phytochromes into fluorescent probes and optogenetic
tools.
PI-083
Luminescent sensor proteins for antibody detection in solution
Remco Arts1, Susann Ludwig1, Marina van Vliembergen1, Vito Thijssen1, Stan van der
Beelen1, Ilona den Hartog1, Stefan Zijlema1, Maarten Merkx1
1Eindhoven University of Technology
Antibody detection is an integral part of many diagnostic strategies, most crucially
so when infectious diseases are involved. Currently used assays, such as ELISA or
SPR, enable detection of antibodies in the laboratory with high sensitivity, yet a
translation of these technologies to an application outside of the laboratory setting
is far from trivial. Problematically, the burden of disease for many infectious diseases
is carried precisely by those countries where access to laboratory facilities is severely
limited. We therefore developed a novel, one-step assay that allows the detection
of antibodies directly in solution using a luminescent sensor protein. Our strategy
is based on the use of a bright luciferase, NanoLuc, tethered to a green fluorescent
protein (mNeonGreen) via a semi-flexible linker containing two epitope sequences.
Crucially, two small helper domains were fused to the protein termini. These domains
keep NanoLuc and mNeonGreen in close proximity in the absence of antibody, enabling
efficient Bioluminescence Resonance Energy Transfer (BRET). Binding of antibody to
the epitopes in the sensor proteins linker domain pulls the BRET partners apart, effectively
changing the color of emission from green to blue. The assay allowed the detection
of picomolar amounts of anti HIV1-p17 antibodies directly in solution, both under
optimized buffer conditions and in blood plasma. In principle. the modular sensor
architecture should allow detection of any antibody with a well-defined epitope of
sufficient affinity. To demonstrate this, the HIV-epitopes were substituted for two
HA-tag epitopes, yielding a sensor that enabled the detection of picomolar amounts
of anti-HA antibodies. The simple optical readout provided by the sensor system allowed
us to record the emitted signal with a conventional mobile phone camera. A simple
software application that analyzes the image based on RGB values sufficed to interpret
the recorded image vis-á-vis the presence of antibody. Bearing in mind the eventually
envisioned application in a point-of-care diagnostic setting, this combination of
sensor recording and interpretation using nothing more than a mobile phone and a software
application holds considerable diagnostic potential. Beyond point-of-care diagnosis
of infectious diseases, a simple assay to detect and quantify antibodies directly
in solution could also have a substantial impact in other fields. Antibodies are ubiquitous
in biotechnology, and this is reflected by the plethora of potential sensor applications,
which range from a role in microfluidic circuits or monitoring the biotechnological
production of antibodies, including validation of bispecificity, to veterinary applications,
diagnosis of autoimmune diseases and monitoring the success of vaccination campaigns.
PI-084
Tertiary Structural Propensities Reveal Fundamental Sequence-Structure Relationships
Fan Zheng1, Jian Zhang2, Gevorg Grigoryan1,2
1Department of Biological Sciences, Dartmouth College, 2Department of Computer Science,
Dartmouth College
The continually growing Protein Data Bank (PDB) has been a key resource for general
principles of protein structure. For example, parsing structural observations in the
PDB into simple geometric descriptors has given rise to statistical energy functions.
Here we present a novel strategy for mining the PDB on the basis of local tertiary
structural motifs (TERM). We define a TERM to be the structural fragment that captures
all local secondary and tertiary structural environments of a given residue, and query
the PDB to obtain quantitative information for each TERMs. First, we show that by
breaking a protein structure into its constituent TERMs, we can describe its sequence-structure
relationship via a new metric we call “structure score.” Using submissions in recent
Critical Assessment of Structure Prediction (CASP) experiments, we find a strong correlation
(R = 0.69) between structure score and model accuracy - a performance that exceeds
leading atomistic statistical energy functions. Next, we show that querying TERMs
affected by point mutations enables the quantitative prediction of mutational free
energies. Our simple approach performs on par with state-of-the-art methods Fold-X
and PoPMuSiC on ∼1300 mutations, and provides superior predictions in certain cases
where other methods tend to fail. In all, our results suggest that the data available
in the PDB are now sufficient to enable the quantification of much more sophisticated
structural observations, such as those associated with entire TERMs, which should
present opportunities for advances in computational structural biology techniques,
including structure prediction and design.
PI-085
Exploiting natural sequence diversity for protein crystallization
Sergio Martínez-Rodríguez1, Valeria Risso1, José M Sanchez-Ruiz1, José A. Gavira2,
1Departamento De Química-Física, Universidad De Granada, 2Laboratorio De Estudios
Cristalográficos, IACT-CSIC-UGR Granada
During the last decade, different rational and high-throughput approaches have been
successfully applied in the protein crystallography field to widen the‖so-called “protein
crystallization bottleneck” [1,2]. Despite the enormous efforts carried out by our
community, the statistics presented by Structural Biology Consortiums [3] suggest
that so far only the easy-to-pick fruit has been attained; thus, new approaches are
necessary to further expand the crystallization limiting step to relevant targets.
On the basis of previous hypothesis suggesting that the difficulties found in protein
crystallization might be a result of evolutionary negative design [4], we have used
two different protein engineering approaches exploiting natural sequence diversity
using beta-lactamase as toolbox: i) ancestral reconstruction and ii) consensus approach
[5]. Both approaches resulted in hyperstable and promiscuous ancestral derivatives.
Furthermore, our initial crystallization results also suggest that both approaches
increased the crystallizability of the resulting enzymes when compared to the extant
TEM-1 beta-lactamase.
[1] Doerr A. Widening the protein crystallization bottleneck. Nat. Methods. 2006.
3:961.
[2] Warke A, Momany C. Addressing the protein crystallization bottleneck by cocrystallization.
Cryst. Growth Des. 2007. 7:2219–2225.
[3] Protein Structure Initiative; http://sbkb.org/
[4] Doye JP, Louis AA, Vendruscolo M. Inhibition of protein crystallization by evolutionary
negative design. Phys Biol. 2004 1:P9-13.
[5] Risso VA, Gavira JA, Mejia-Carmona DF, Gaucher EA, Sanchez-Ruiz JM. Hyperstability
and substrate promiscuity in laboratory resurrections of precambrian β-lactamases.
J Am Chem Soc. 2013. 135:2899-2902.
PI-086
Synthesis of selectively functionalized adiponectin
Andreas Mattern1, Annette Beck-Sickinger1
1University of Leipzig, Institute of Biochemistry
The adipocyte-derived hormone adiponectin has become a key player for the understanding
of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic
syndrome. One of its major functions are the insulin sensitizing effects, which are
mediated by the activation of AMPK, p38-MAPK and PPARα (1). Furthermore adiponectin
is involved into glucose regulation and fatty acid oxidation. Recently, three adiponectin
receptors AdipoR1, AdipoR2 and T- cadherin have been described while an unknown fourth
receptor is hypothesized (2). For only two of them (AdipoR1 and AdipoR2) the signaling
transduction via adiponectin has been confirmed (3). In order to find new binding
partners or co-receptors, we cloned and expressed full length adiponectin as a fusion
protein with a C-terminal intein and a chitin binding domain (CBD) as well as an N-terminal
His10-tag. By using the IMPACT-system, the fusion protein was cleaved to form the
corresponding thioester. To separate the starting materials as well as the cleaved
intein chitin binding domain, the purification was performed with chitin beads. Furthermore,
the product was concentrated by Ni-NTA-affinity chromatography. Accordingly, the obtained
adiponectin thioester was reacted with a TAMRA- or a biotin labeled peptide, respectively,
to receive the corresponding ligation product. Finally the functionalized adiponectin
was purified by size exclusion chromatography. Further studies will allow screening
for interacting molecules in cell and tissue derived samples. (1) Hui et al. (2012)
British Journal of Pharmacology 165, 574–590. (2) Awazawa et al. (2011) Cell Metabolism
13, 401–412. (3) Heiker et al. (2010) Biol. Chemistry 39, 1005-1018.
PI-087
De novo catalysis in ancestral protein scaffolds
Valeria A. Risso1, Sergio Martinez-Rodriguez1, Adela M. Candel1, David Pantoja-Uceda2,
Mariano Ortega-Muñoz3, Francisco Santoyo-Gonzalez3, Marta Bruix2, José A Gavira4,
Jose M. Sanchez-Ruiz1
1Departamento de Quimica Fisica, Facultad de Ciencias University of Granada, 2Dpto.
de Quimica Fisica Biologica. Instituto de Quimica Fisica Rocasolano, 3Departamento
de Quimica Organica, Facultad de Ciencias University of Granada, 4Laboratorio de Estrudios
Cristalograficos, IACT-CSIC-UGR Granada
Rational design of non-natural enzyme activities has proved challenging. Here, we
report the introduction of catalysis of the Kemp elimination (a model of proton abstraction
from carbon) in scaffolds corresponding to Precambrian nodes in the evolution of the
antibiotic resistance protein β-lactamase. We used a single-mutation, minimalist approach
based on chemical intuition, and obtained catalysis levels similar to those reported
in the literature for computational Kemp-eliminase designs involving multiple mutations.
Remarkably, the approach was unsuccessful when performed on modern β-lactamases. We
provide experimental evidence that enhanced conformational flexibility contributes
to the success of the minimalist design in the ancestral scaffolds. This work has
implications for the understanding of function emergence in protein evolution and
demonstrates the potential of ancestral protein resurrection in enzyme engineering
and design.
PI-088
Exploring the Importance of Dimerization for DJ-1 Function through Engineered Domain
Fusions
Sierra Hansen1, Jiusheng Lin1, Mark Wilson1
1University of Nebraska
Parkinson’s Disease is a progressive neurodegenerative disease that affects approximately
6.3 million people worldwide and is characterized by the loss of dopaminergic neurons
in the substantia nigra pars compacta. DJ-1 (PARK7) is one of several genes that are
mutated in rare forms of familial parkinsonism. DJ-1 is a dimeric cytoprotective protein
that defends against oxidative stress and preserves mitochondrial function. Dimerization
of DJ-1 is thought to be essential for this function, as some disease-associated mutations
cause poor folding and disrupt the DJ-1 dimer. However, recent reports suggest that
DJ-1 may be functional as a monomer. To test this, we have engineered a non-dissociable
DJ-1 dimer that is a fusion of two human DJ-1 domains. This construct cannot dissociate
into monomers and thus will provide a stringent test of the importance of monomeric
DJ-1. Our engineered construct is modeled on plant DJ-1 homologs, which feature naturally
occurring duplicate DJ-1 domains separated by a small (19 amino acid) linker region.
Using X-ray crystallography, we confirmed that this engineered non-dissociable human
DJ-1 dimer has identical structure to the naturally occurring dimeric protein. We
have investigated the influence of enforced dimerization of the pathogenic effects
of the parkinsonian L166P and L10P mutations. CD spectroscopic analysis reveals that
single and double L166P mutations in the non-dissociable DJ-1 dimer maintain a higher
degree of structure than L166P mutations in the native protein. Additional characterization
of the protective capacity and subcellular trafficking of this non-dissociable DJ-1
dimer is underway.
PI-089
The purification, crystallization and preliminary characterization of SdrE from S.
aureus
Deqiang Wang1, Ke Chen1, Jun Zhang2
1Key Laboratory of Molecular Biology on Infectious Disease, 2The Department of Cell
Biology and Genetics
The purification, crystallization and preliminary characterization of SdrE from S.
aureus Staphylococcus aureus (S.aureus) is an important human opportunistic pathogen
which colonizes about 20% of the human population persistently [1]. Surface proteins
of S.aureus can excretion a kind of sortase, which represents a surface organelle
responsible during the pathogenesis of bacterial infection the host circulation [2].
Sdr proteins were a component of cell wall anchored family proteins, including SdrC,
SdrD and SdrE [3]. SdrE could combine with the complement regulatory protein factor
H to escape the alternative pathway of complement [4]. To further investigate the
functions of SdrE, we have expressed and purified the adhesive domain (residues 141-’06:15),
and crystallized the recombinant protein. In addition, we also constructed the mutant
S.aureus, and the cell experiments confirmed that SdrE gene participate in the bacteria
invasion.
Refereces:
[1] Miller LS., et al., Nature Reviews 2011,8,505-518;
[2] Brien LO., et al., Molecular Microbiology, 2002, 44:1033-1044;
[3] Sitkiewicz I., et al., Antonie van Leeuwenhoek, 2011, 99:409-416; [4] Sharp JA.,
et al., PLoS One, 2012,7(5):e38407
PI-090
Structure based modifications of the bacterial microcompartment shell protein PduA
David Leibly1,2, Julien Jorda2, Sunny Chun3, Alan Pang2, Michael Sawaya2, Todd Yeates1,2,3
1Department of Chemistry and Biochemistry, University of California, 2UCLA-DOE Institute
for Genomics and Proteomics, 3Molecular Biology Institute, University of California
Bacterial Microcompartments (BMCs) are proteinaceous organelles that sequester key
metabolic reactions to increase enzymatic efficiency or to prevent the loss of volatile
or toxic intermediates. There is an increasing desire to engineer BMCs for non-native
enzymatic processes. It is thought this will increase multi-enzyme pathway efficiency
and allow the expression pathways that may produce toxic or volatile intermediates
in bacteria. The mechanisms of small molecule transport and retention of toxic intermediates
by BMCs remain poorly understood. Better understanding of the BMCs pores critical
to engineer BMCs for these non-native pathways. In order to better understand the
BMC pore we have undertaken structure-guided modifications of the the hexameric PduA
shell protein of the 1,2-propanediol utilization microcompartment (Pdu MCP). These
modifications include pore mutations in an attempt to alter substrate specificity
and permutations of PduA to allow more drastic alterations to the structure of the
protein. Crystal structures of PduA pore mutants, solved to atomic resolution (2-3.3Å)
provide evidence of the pore residues that confer specificity. Further, a PduA permutation
(PduAp) has resulted in a closed icosahedral cage. This novel PduAp cage shows a pH
and salt dependent assembly and may serve as a reaction vessel or be utilized for
cargo delivery.
PI-091
Targeted conformational transitions of large and multimeric proteins by an efficient
elastic network based technique
Yasemin Yesiltepe1, Doga Findik1, Arzu Uyar1, Deniz Turgut2, Rahmi Ozisik2, Turkan
Haliloglu1, Pemra Doruker1
1Bogazici University and Polymer Research Center, 2Rensselaer Polytechnic Institute
ANM–MC is a computationally efficient, coarse-grained simulation technique that integrates
anisotropic network model (ANM) with knowledge-based Monte Carlo (MC) energy minimization
method (1, 2). ANM–MC is used to identify targeted transition pathways and intermediates
between open and closed states of proteins. At each step of this iterative technique,
the protein is deformed along the collective ANM mode showing the best overlap with
the target direction and its energy is minimized via short MC run. In this work, optimization
of simulation parameters (number of MC moves and their perturbation strength, ANM
deformation factor in each cycle and force constant for backbone bonds) was performed
in order to increase the efficiency of this technique. As a result, this technique
can now be applied to much larger systems and conformational changes. The transition
pathway between apo and DNA-bound conformations of the yeast RNA polymerase, which
is a hetero-10-mer with more than 3500 residues, will be presented here. Moreover,
the pathway intermediates for more than 10 diverse proteins were analyzed in terms
of changes in local strain energy and backbone torsional angles during apo-to-complex
transitions. Certain residues interacting with the ligand are detected to exhibit
large changes with respect to any of these two parameters for more than half of the
proteins in our dataset.
References:
1. Kantarci-Carsibasi, N., T. Haliloglu, and P. Doruker. 2008. Conformational Transition
Pathways Explored by Monte Carlo Simulation Integrated with Collective Modes. Biophys.
J. 95:5862–5873.
2. Uyar, A., N. Kantarci-Carsibasi, T. Haliloglu, and P. Doruker. 2014. Features of
Large Hinge-Bending Conformational Transitions. Prediction of Closed Structure from
Open State. Biophys. J. 106:2656–2666.
PI-092
Continuous directed evolution of receptor-selective α-endotoxins for overcoming insecticidal
resistance
Ahmed Badran1,2, Victor Guzov3, Qing Huai3, Melissa Kemp3, Prashanth Vishwanath3,
Artem Evdokimov3, Farhad Moshiri3, Meiying Zheng3, Keith Turner3, David Liu1,2
1Department of Chemistry and Chemical Biology, Harvard University, 2Howard Hughes
Medical Institute, Harvard University, 3Monsanto Company
Transgenic crops have radically reshaped the agricultural landscape. Since their introduction
in the late 1990s, transgenic crops have affected economic gains greater than US$110
billion globally due to reduced production costs and increased yield gains. Crops
modified to produce biological insecticides derived from the soil bacterium Bacillus
thuringiensis (Bt) are among the most robust methods of pest control. Bt toxins offer
many advantages over traditional insecticides, chiefly their inability to affect human
biology and exquisite selectivity for defined pest species. However, the evolution
of resistance to Bacillus thuringiensis ∂-endotoxins (Bt toxins) in insects has been
widely observed in the field, and greatly threatens the use of this mechanism of pest
control in the future. We developed a Phage-Assisted Continuous Evolution (PACE) platform
for the rapid generation of high-affinity protein-protein interactions and validated
the system by evolving known high affinity antibody mimetics in <5 days of PACE. We
applied this system to the evolution of the Bt toxin protein Cry1Ac to recognize a
non-cognate cadherin-like receptor from Trichoplusia ni, a pest for which Bt toxin
resistance has been observed in both the laboratory and the field. The resulting evolved
Cry1Ac variants exhibits high affinity for the target receptor, and kill insect cells
more potently than wild-type Cry1Ac. Our findings establish that the directed evolution
of novel receptor recognition in Bt toxins can be used to target resistant pests,
and has far-reaching implications for biological reagents and therapeutics.
PI-093
Optimization of a designed protein-protein interface
Brian Maniaci1, Collin Lipper2, John J. Love1
1San Diego State University, 2University of California
Protein-protein interactions play key roles in practically every biological process.
Protein-protein interactions vary with composition, affinity, and lifetime of the
complex. Studying designed protein-protein interactions will provide insight into
the underlying principles of complex assembly and formation. Computational protein
docking and amino acid sequence design were used previously to generate protein dimers
from monomeric proteins. The normally monomeric β1 domain of Streptococcal protein-G
(GB1) was computational docked to itself, followed by optimization of the interfacial
side chains. Two variants, MonomerA and MonomerB, were computationally derived as
a result of a designed protein-protein interface. These designed proteins were characterized
using analytical ultracentrifugation and heteronuclear NMR techniques. This design
resulted in a pair of protein monomers that formed a heterodimer of modest binding
affinity. A tetrahedral metal-templated interface design strategy was implemented
in an attempt to strengthen the MonomerA-MonomerB complex by introducing cross-monomer
metal coordination. Another advantage of using the metal-templated interface is the
ability to control the protein-protein interaction both temporarily and spatially.
A number of newly engineered variants of Monomer A and Monomer B with metal coordination
sites were designed, produced, and tested for increased affinity of the protein-protein
complex. While the generation of a metal-templated MonomerA-MonomerB complex was unsuccessful,
we were able to obtain MonomerA variants that form a homodimer assembly only in the
presence of Zinc (II) ions. The crystal structures of metal-templated MonomerA variants
in the presence of zinc provide an explanation for the observed dimer formation. The
crystal structure indicates that the protein-protein interaction is not driven by
the designed protein interface, but rather non-specific association via edge-strand
interactions. New variants were designed with the goal of engineering a high affinity
homodimer in a helix-to-helix orientation as the originally designed protein-protein
interface. Current evaluation of MonomerA variants for self-association via metal
coordination are being evaluated using size exclusion chromatography with a multi-angle
light scattering detector for oligomerization state quantification. The results of
this protein design project should lead to a greater understanding of the biophysical
parameters that drive natural protein-protein interactions.
PI-094
Continuous evolution of site-specific recombinases with highly reprogrammed dna specificities
Jeffrey L Bessen1,2, David B Thompson1,2, David R. Liu1,2
1Department of Chemistry & Chemical Biology, Harvard University, 2Howard Hughes Medical
Institute, Harvard University
The ability to precisely modify the genome of human cells has enormous potential as
a novel therapy and a powerful research tool. In contrast to reprogrammable nucleases,
such as TALENs or a Cas9/sgRNA pair – which specifically cleave DNA but then rely
on stochastic host cells processes to effect gene insertion – site specific recombinases
directly catalyze genomic integration with high efficiency. A major limitation of
this approach is that recombinases, such as Cre, natively bind with high specificity
to long DNA target sequences (LoxP in the case of Cre) that do not exist in the human
genome. Previous attempts at evolving Cre resulted in modest changes to its specificity,
or required hundreds of rounds of manual protein evolution. We developed and validated
a Phage Assisted Continuous Evoluiton (PACE) selection for rapidly altering the DNA
specificity of Cre recombinase towards a site present in a human genomic safe harbor
locus. The PACE experiments resulted in Cre variants capable of recombining a substrate
with nearly 50% of the nucleotides altered compared to LoxP. We successfully used
one of these variants to integrate exogenous DNA into the genome of unmodified human
cells. We are currently using sequencing methods to determine the specificity of the
new recombinase clones.
PI-095
Generation of comprehensive deletion libraries mediated by in vitro transposition
Aleardo Morelli1, Burckhard Seelig1
1University of Minnesota
Generation of comprehensive deletion libraries mediated by in vitro transposition
Analysis of protein enzymes and ribozymes from nature, and from in vitro evolution,
revealed that deletions of up to dozens of amino acids (or nucleotides) can be structurally
tolerated. Furthermore, shortened variants can exhibit better stability and increased
catalytic activity. In order to investigate the effects of deletions, we developed
a new procedure based on in vitro transposition to build libraries of more than 10,000
deletion mutants in three to four days. We tested our procedure on DNA sequences coding
for an artificial RNA ligase called ligase 10C. We used the generated library for
an mRNA display selection, and isolated two active mutants containing 18 and 13 amino
acids N-terminal deletions.
PI-096
Structural characterization of PpsC, a multi-domain polyketide synthase from Mycobacterium
tuberculosis using a fragment-based approach
Alexandre Faille1, Nawel Slama11, Anna Grabowska1, David Ricard1, Annaik Quémard1,
Lionel Mourey1, Jean-Denis Pedelacq1
1Institut de Pharmacologie et de Biologie Structurale
Polyketide synthases are of great interest in numerous scientific fields. They are
composed by multiple domains, each having a different role to play in the catalysis
of sequential reactions including condensation, reduction and esterification. Their
reaction products, named polyketides, represent a large variety of chemical compounds,
from antibiotics to immunosuppressors or even anticancer drugs. PpsC is a 231 KDa
polyketide synthase, organised into six catalytic domains (KS-AT-DH-ER-KR-ACP) with
singular functions. Along with other type I polyketide synthases, PpsC is responsible
for the biosynthesis of an essential polyketide for the virulence of Mycobacterium
tuberculosis (Mtb) and thus is a target of choice for the design of inhibitors. To
date, no structural information of any type I Polyketide synthase in its entire form
has been described. Main reasons are the length of these large size enzymes and the
flexibility imposed by the linkers between domains, thus making them very difficult
to crystallize. Numerous questions about domain-domain interactions, spatial arrangement
of this complex machinery, substrate specificity and stereochemistry are still unanswered.
Addressing the structural and functional characterization of PpsC would then help
answering these questions and provide valuable information for drug design. To overcome
the length- and flexible- dependent problem originating from the presence of multiple
domains and linkers, we decided to study domains expressed alone. For this purpose,
we used our domain trapping strategy to identify soluble fragments representing a
single domain from PpsC [1]. It has the advantage of not relying on the bioinformatically
designed domain boundaries and can even sometimes include parts of linkers to obtain
more soluble fragments. Using this strategy, we were able to identify relatively small
and highly soluble fragments representing each domain of PpsC, thus facilitating the
downstream structural and functional characterization. More than 20 fragments have
been submitted to crystallization trials. Among these, 5 gave crystals and allowed
us to determine the X-ray structure of PpsC AT, ER, in addition to the DH domain in
complex with a substrate analog for which activity was confirmed in vitro.
[1] J.D. Pedelacq et al. Experimental mapping of soluble protein domains using a hierarchical
approach. Nucleic Acids Res. 2011 October; 39(18): e125.
PI-097
Computational design of tighter protein-ligand interfaces
Brittany Allison1, Brian Bender2, Jens Meiler1,2
1Vanderbilt University, Department of Chemistry, 2Vanderbilt University, Department
of Pharmacology
The computational design of proteins that bind small molecules remains a difficult
challenge in protein engineering. The ability to computationally design native-like
interactions with high accuracy and efficiency would be an asset towards therapeutic
development, enzyme design, and engineering functional proteins. We have developed
a systematic approach to designing interfaces. We first identify ligands with naive
binding affinity to our protein scaffold, then use RosettaLigand to computationally
dock the ligand while designing the interface for a tighter interaction. This way,
we are taking a ‘shot in dim light’ for design as opposed to a ‘shot in the dark’,
allowing us to more thoroughly investigate the successful and not-so-successful designs,
and improve the computational methods. Of ∼3500 ligands screened, we identified 28
weakly-binding hits in the range of 340 – 1110 µM. Thus far, RosettaLigand has successfully
designed one tighter protein-ligand interface, from 312 µM to 21 µM. In progress experiments
include designing and experimentally validating more designed interfaces.
PI-098
Structural studies of human acidic fibroblast-growth factor (FGF1) mutants with a
probable anticancer activity
Maria Cecilia Gonzalez1, Stefano Capaldi1, Maria Elena Carrizo1, Laura Destefanis1,
Michele Bovi1, Massimiliano Perduca1, Hugo Luis Monaco1
1Biocristallography Laboratory, Department of Biotechnology, University of Verona
Lectins are carbohydrate-binding proteins ubiquitously present in nature. They play
a role in biological recognition phenomena involving cells and proteins. The interaction
lectin-carbohydrate is highly specific, and can be exploited for the development of
nanoparticles containing on their surface lectins specifically directed to carbohydrate
residues present only on malignant cells and absent on healthy ones (1). Lectins have
been found to possess anticancer properties and they are proposed as therapeutic agents,
binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis
and inhibition of tumor growth. Some lectins are able to prevent the proliferation
of malignant tumor cells because they recognize the T-antigen (Gal β 1–3GalNAc) found
specifically on the surface of tumor cells (2). The main problem is that their use
as a detection agent for the T-antigen in clinical studies is not possible because
the immune system can recognize them as foreign molecules and develop an immune response.
Previous studies with X-ray crystallography made in our laboratory have characterized
a lectin found in mushrooms called BEL β-trefoil which has antiproliferative activity
on tumor cell lines, because it contains three binding sites for the T-antigen. Unlike
other lectins with this property, BEL β-trefoil shows structural homology with a human
protein, acidic Fibroblast Growth Factor (FGF1) (3). Superposition of their structures
suggests that the human protein could be mutated to contain at least one of the binding
sites for the T-antigen. Such mutations should create in FGF1 the potential capacity
of recognizing tumor cells with less immunogenicity than the fungal protein. FGF1
is mitogenic and chemotactic, and mediates cellular functions by binding to transmembrane
receptors, which are activated by ligand-induced dimerization requiring heparin as
co-receptor. To reach our purpose, the FGF1 cDNA was cloned into a bacterial plasmid
and then mutated in five different positions to eliminate its mitogenic activity and
to engineer in the protein the T-antigen binding capacity. Attempts to crystalize
the mutants of FGF1 were made using the hanging drop technique with the final aim
to carry out their structural characterization by X-ray diffraction analysis of the
crystals.
References:
[1] Lis H and Sharon N. Lectins as molecules and as tools. Annu Rev Biochem. 1986.
55(1): p. 35-67.
[2] Ju T, Otto VI, Cummings RD. The Tn antigen-structural simplicity and biological
complexity. Angew Chem Int Ed Engl. 2011. 50(8): p.1770-1791.
[3] Bovi M, Cenci L, Perduca M, Capaldi S, Carrizo ME, Civiero L, Chiarelli LR, Galliano
M, Monaco HL. BEL β-trefoil: a novel lectin with antineoplastic properties in king
bolete (Boletus edulis) mushrooms. 2013. Glycobiology. 23(5): p. 578-592.
PI-099
Drug-controllable protein tags for the selective visualization or selective shutoff
of newly synthesized proteins of interest in mammalian cells and in vivo
Conor Jacobs1, Yang Geng2, Ryan Badiee1, Tiffany Nguyen3, Andrew Evans4, Hokyung Chung1,
Ying Yang2, Mehrdad Shamloo4, Roger Y. Tsien5, Michael Z. Lin2,6
1Department of Biology, Stanford University, 2Department of Pediatrics, Stanford University,
3Department of Neurology and Neurological Sciences, Stanford University, 4Department
of Neurosurgery, Stanford University, 5Department of Pharmacology, UC San Diego, 6Department
of Bioengineering, Stanford University
The de novo synthesis of proteins in response to the activation of cellular signaling
pathways is a crucial element of many high-level biological processes, including the
synaptic plasticity underpinning memory formation in the brain. While of fundamental
biological importance, there has been a shortage of tools with which to specifically
target pools of newly synthesized proteins of interest for study. Thus, we have developed
TimeSTAMP and SMASh, methods for drug-dependent tagging, or destruction, respectively,
of newly synthesized copies of proteins of interest. Both methods rely on protein
tags that remove themselves by default via an internal Hepatitis C Virus (HCV) NS3
protease, but which are retained in the presence of cell-permeable small molecule
protease inhibitors. The TimeSTAMP tag contains split YFP halves and epitope tags
which are reconstituted and preserved, respectively, on proteins of interest following
drug application, whereas the SMASh tag contains a strong degron which remains attached
to proteins of interest following drug application, resulting in their clearance.
One limitation of TimeSTAMP and SMASh is that they can only be used to independently
manipulate one protein of interest at a time. Furthermore, the application of TimeSTAMP
and SMASh to study endogenous protein pools in mammals has not yet been explored.
Here, we report on efforts to extend these techniques by reengineering NS3 proteases
which can be inhibited by two different drugs orthogonally to one another. By incorporating
different drug resistance mutations into two NS3 protease variants, we engineered
NS3 protease domains that are inhibitable either by asunaprevir only, or by telaprevir
only. We found that these tags permit simultaneous and independent control over the
newly synthesized pools of two proteins of interest within the same population of
cells. We also report the development of transgenic knock-in mouse strains incorporating
TimeSTAMP and SMASh tags, which allow the interrogation of newly synthesized pools
of specific endogenous synaptic proteins in the context of their endogenous regulatory
elements, and without relying on overexpression.
PI-100
BRET-based protein switches for detection of Dengue serotype 1 antibodies
Remco Arts1, Susann Ludwig1, Byron Martina2, Maarten Merkx1
1Eindhoven University of Technology, 2Erasmus Medical Center Rotterdam
Infectious diseases are often diagnosed by the presence of specific antibodies that
are produced in response to the invading pathogen. One example are antibodies that
are present in patient blood after infection with the Dengue virus serotype 1 and
that are directed against an epitope on the virus’ non-structural protein 1 (NS-1).
Traditional antibody diagnosis relies on time-consuming multi-step assays that require
sophisticated equipment in a laboratory environment. A promising alternative are protein
switches that are based on bioluminescence resonance energy transfer (BRET). These
switches comprise a luciferase (NanoLuc) and a green fluorescent protein (mNeonGreen),
which are connected via a semi-flexible linker. The linker contains two epitope sequences
of NS-1 to which the antibodies bind specifically. If no antibodies are present NanoLuc
and mNeonGreen are held in close proximity via two helper domains and BRET can occur;
thus green light originating from mNeonGreen is visible. If antibodies are present,
they bind to the specific epitopes in the linker of the switch and cause stretching
of the linker and therewith break the interaction of the helper domains. As a result,
NanoLuc and mNeonGreen are separated in such a way that BRET cannot occur anymore;
thus only blue light originating from NanoLuc remains visible. Using this principle,
monoclonal anti-NS-1 antibodies were detectable in a controlled buffer system and
in spiked plasma samples. Furthermore, the developed antibody switch was applied to
plasma samples of macaques after a primary infection with Dengue virus serotype 1.
Signal readout was possible using a laboratory-based plate reader as well as the camera
of a standard smartphone. We demonstrate that this BRET-based protein switch can quickly
detect antibodies in solution in a single-step assay format using simple equipment
for signal readout, such as a standard smartphone. This simplified antibody detection
platform has the potential to be carried out outside of a laboratory, thus in areas
with limited laboratory infrastructure and a high number of diverse infectious diseases.
PI-101
Delivery of biologics against intracellular targets
Paulina Kolasinska-Zwierz1, Pawel Stocki1, Bina Mistry1, Sandrine Guillard1, Alison
Smith1, Rose Marwood1, Ben Kemp1, Anna Czyz1, Ronald Jackson1, Ralph Minter1, Tristan
Vaughan1, Herren Wu1
1ADPE Cambridge, MedImmune, Milstein Building
Proteins expressed from more than two-thirds of the human genome reside within intracellular
compartments. Of these proteins many are important disease-related targets such as
KRas and c-Myc which cannot be easily addressed by conventional small molecule approaches.
Some of the weaknesses of small molecules can be addressed by biologic drugs, for
example high target specificity and inhibition of protein-protein interactions. The
challenge for biologics is how to engineer recombinant proteins to access the intracellular
space. One strategy is to use systems evolved by bacteria and viruses to deliver material
inside the cells. An example of such pathway is used by Pseudomonas Exotoxin A (PE).
The modularity of PE allows the catalytic domain to be replaced with a biologic payload
against desired intracellular target. An additional benefit of PE-based delivery is
a possibility of targeting the drugs only to relevant cells in the body by modifying
the cell-targeting domain of the PE. The aim of this project is to deliver functional
payloads against K-Ras and c-Myc into the cell using a Pseudomonas Exotoxin A translocation
domain. We used phage and ribosome display to select antibody mimetics that bind K-Ras
and c-Myc. Here, we present their activity in biochemical assays and the initial results
on generation of PE-based constructs.
PI-102
Recombinant H5 antigen based on hydrolytic domain with deletion of polybasic cleavage
site forms functional oligomers
Edyta Kopera1, Maria Pietrzak1, Agnieszka Macioła 1, Anna Maria Protas-Klukowska1,
Konrad Zdanowski1, Beata Gromadzka2, Krystyna Grzelak1, Zenon Minta3, Krzysztof Śmietanka3,
Bogusław Szewczyk2
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 2University
of Gdansk and Medical University of Gdansk, 3National Veterinary Research Institute,
Department of Poultry Diseases
Influenza virus hemagglutinin (HA) is a glycoprotein studded in the lipid bilayer
of the virus (1). Hemagglutinin is synthesized as HA0 molecule assembled as noncovalently
bound homotrimers on the viral surface. This precursor protein is cleaved by trypsin-like
proteases to yield two subunits HA1 and HA2 linked by a single disulphide bond (2).
HA0 is also post-translationally modified by N-glycosylation (3). It is well established
that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies.
In the attempt towards developing influenza vaccine production (the egg-based manufacturing
lasts several months) that would be faster and safer the utilization of recombinant
antigen alone is currently being observed. Recently we demonstrated that yeast produced
influenza H5 protein although cleaved into two subunits induced strong immunological
response in mice (4). In this report, we describe the biochemical and immunological
characterization of the H5 antigen, based on hydrolytic domain of the H5N1 gene, with
deletion of multibasic cleavage site and expressed in yeast system. The HA encoding
gene from H5N1 virus with deletion of 18 nucleotides was cloned into pPICZαC vector.
rHA fusion protein with His6-tag was secreted into the culture medium and was purified
to homogeneity in one step using Ni-NTA agarose. The efficiency of the antigen purification
was 200 mg/L. Glycosylation sites of rHA were determined using LC-MS-MS/MS. Analysis
of the N-linked glycans revealed that the rHA is glycosylated at the same sites as
the native HA in the vaccine strain. Next we investigated if the hemagglutinin with
deletion of the cleavage site oligomerize into higher molecular forms. To determine
the oligomeric forms of the recombinant antigen various approaches were applied e.g.
Native-Page, Size Exclusion Chromatography or Dynamic Light Scattering. As a final
experiment to measure the size of oligomers in a protein sample a combined technology
SEC-MALS was conducted, using multi angle light scattering (MALS) as a detector. The
immunological activity of rHA was tested in chicken and mice, where antigen elicited
high immune response. The data presented here demonstrate that new influenza antigen
produced in P. pastoris is highly immunogenic and might be consider as a candidate
for subunit vaccine.
Bibliography
1. Yin J.et al. Virologica Sinica. 2013, Vol. 28, 1, pp. 003-015.
2. Skehel J.J., Wiley D.C. Annu Rev Biochem; 2000, Vol. 69:531-69.
3. Grinna L.S., Tschopp J.F. s.l.: Yeas, 1989, Vols. 5:107-’01:15.
4. Kopera E et al. Acta Biochimica Polonica. 2014, Vol. 64, pp. 597-602.
PI-104
Mining the structural universe for de novo design
Craig Mackenzie1, Gevorg Grigoryan1
1Dartmouth College
Structural motifs capture redundant patterns that frequently occur in proteins. Motifs
associated with contiguous fragments of structure (i.e., secondary structural motifs)
are well studied and have been successfully used to capture “rules” describing sequence-structure
relationships in protein design and structure prediction. We have extended this concept
to motifs that capture tertiary information–(i.e., tertiary structural motifs or TERMs.
We have discovered that a relatively small alphabet of TERMs describes the known structural
universe (all secondary, tertiary and quaternary information in the PDB) at sub-Angstrom
resolution. This alphabet of universal motifs reveals the remarkable degeneracy of
the protein structure space, with just a few hundred TERMs sufficient to accurately
capture half of the known structural universe. We have begun to demonstrate the considerable
promise this structural alphabet has for applications such as protein design, structure
prediction, and docking. We have developed a novel protein design framework that selects
amino acid sequences, given a desired structure, using solely information from the
universal TERMs. We show that given a native backbone, this framework recovers the
native sequences to a level on par with state-of-the-art atomistic protein design
methods, indicating that the motifs capture the salient structural rules governing
native proteins. Further, predicted sequence distributions agree closely with observed
evolutionary variation. Given the apparently high degeneracy among even complex features
of protein structure, methods based on mining the PDB for tertiary information should
provide ample opportunities for advancement in problems of computational structural
biology.
PI-106
Sortase-mediated synthesis of protein-DNA conjugates for sensitive biosensing
Bedabrata Saha1, Marieke op de Beeck1, Remco Arts1, Maarten Merkx1
1Department of Biomedical Engineering, Eindhoven University of Technology
In recent years, semisynthetic protein-DNA conjugates have emerged as attractive biomacromolecules
for different applications in bio-nanotechnology, biosensing, diagnostics and therapeutics.
In protein-DNA conjugates, synthetic oligonucleotides allow the construction of desired
molecular architecture with high specificity, while maintaining the original functionality
of the protein molecules for desired application. However, the synthesis of site-specific
and stoichiometric protein-DNA conjugates can be challenging. Due to the diversity
in composition and physico-chemical properties of the proteins, few generic strategies
are available for conjugation of protein molecules to a DNA scaffold. A common approach
is to use thiol-based covalent conjugation, but the introduction of additional cysteines
can lead to the formation of intermolecular disulfides or interfere with the formation
of native disulfide bonds. As an alternative, here we have developed a site-directed
protein-DNA conjugation strategy based on sortase mediated trans-peptidation reaction.
The sortase recognizes a ‘sorting motif’ (i.e. LPXTG, X any amino acid), which is
recombinantly introduced by site-directed mutagenesis at the C-terminal end of the
protein molecule. The sortase cleaves the T-G peptide bond and catalyzed the formation
of a new amide bond between the LPXT peptide and the N-terminal amine of any molecule
bearing an N-terminal oligoglycine motif. For this purpose, a triglycine motif was
introduced at the 5’-end of single-stranded DNA (ssDNA). On-column synthesis of triglycine
modified ssDNA, protected on a controlled pore glass beads, simplified the purification
process and enhanced the yield of triglycine-modified ssDNA (> 90%). We used this
conjugation strategy in several biosensing applications. For example, we used the
method to conjugate ssDNA linkers at the C-termini of a range of single-chain antibody
fragments (scFv) and applied these constructs to allow oriented display of capture
molecules on biosensor surfaces. ssDNA-scFv were Using an excess of triglycine modified
ssDNA, we achieved ∼55% conversion scFv-ssDNA conjugate, which can be further purified
by in two step purification process consisting of Ni-NTA affinity column and ion-exchange
chromatography. We also extended this sortase-based conjugation strategy to develop
a bioluminescence based assay for sensitive target oligonucleotide detection. In this
regard, the 5’ and 3’ end triglycine-modified ssDNA molecules were successfully conjugated
with a BRET protein pairs, NanoLuc luciferase and mNeonGreen fluorescent protein.
The introduction of a C-terminal sortase-His5 tag and and N-terminal Strep–tag allowed
efficient purification of theseprotein-ssDNA conjugates from excess oligonucleotides
and unreacted protein.
PJ-001
Mass spectrometry based proteomics to identify the protein differences in human breast
milk from breast cancer patients and controls
Devika Channaveerappa1, Roshanak Aslebagh1, Kathleen F. Arcaro2, Costel C. Darie1
1Clarkson University, 2University of Massachusetts
Breast cancer is the second leading cause of cancer death in women. About 12% women
in the US develop breast cancer. Death rates due to breast cancer have been declined
over the years due to advancements in mammography and treatment. Although, mammography
helps in the early detection of breast cancer, it has few limitations. Dense breast
tissue makes mammogram less accurate. Breast milk can be assessed to evaluate the
risk of one getting breast cancer by comparing the proteomes of breast milk from healthy
and breast cancer suffering individual. This study makes use of mass spectrometry
based proteomics to identify the differences between the control and cancerous samples
which would further help in identifying potential biomarkers for breast cancer. Firstly,
SDS-PAGE was used to separate the proteins from the whole milk sample. The gel bands
for each sample was then excised and cut into small pieces. The gel pieces were washed
and trypsin digested in order to extract the peptides. Peptide mixtures in the solution
were cleaned using C18 Zip-tipp and then analyzed by liquid chromatography-tandem
mass spectrometry (LC- MS/MS). 60 minutes and 120 minutes gradient were used for LC-MS/MS
analysis. Raw data obtained were converted to pkl files using ProteinLynx Global Served
(PLGS version 2.4). Raw data were then submitted to Mascot database search for protein
identification. The Mascot results were then exported as .dat files and further analyzed
using Scaffold version 4.1 software. Three breast cancer milk samples were investigated
against healthy control milk samples. In the SDS-PAGE gel, after Coomassie staining,
the protein patterns did show minor differences. After LC-MS/MS analysis, the proteins
identified by Mascot database search were imported into the Scaffold software and
compared for the relative ratio between the proteins from the milk sampled from control
donors and the donors with breast cancer. There were significant differences identified
in the proteomes of the two sets of samples. Some of the proteins were upregulated
in the breast cancer samples and some were down regulated when compared with the controls.
Additional investigation of more breast milk samples is ongoing. This study focuses
on identifying biomarkers directly in the milk of donors with breast cancer.
PJ-002
Leukolike Vectors: leukocyte-inspired nanoparticles
Claudia Corbo1,2, Alessandro Parodi1,2, Roberto Palomba1,2, Roberto Molinaro1, Michael
Evangelopoulos1, Francesco Salvatore2,3, Ennio Tasciotti1
1The Houston Methodist Research Institute, 2Fondazione IRCCS SDN, 3CEINGE-, Biotecnologie
Avanzate
Nanomedicine aims to improve drug efficiency by enhancing targeting and biocompatibility,
and reducing side effects. Multiple surface modifications have been proposed to provide
nanocarriers with these features, based on complex synthesis processes and very often
inefficient in contemporary providing biological tolerance and targeting properties
[1]. Bio-inspired approaches based on surface coatings developed from the purified
cell membrane of immune cells represents a new paradigm shift for the development
of carrier enable of prolong circulation and proper tumoritropic capabilities. We
showed that nanoporous silicon (NPS) particles coated with leukocyte cellular membranes
-Leukolike Vectors (LLVs) – possess cell-like properties [2]. LLVs can escape macrophage
uptake, delay sequestration by the reticulo-endothelial system, target tumor inflamed
vasculature and accumulate within the cancer parenchyma [2]. LLVs were fully characterized
for their shape, size, surface charge and coating through dynamic light scattering
and scanning electron microscopy. In addition we characterized the content and function
of the leukocyte’s proteins transferred onto the LLVs coating through high-throughput
proteomic analysis and the results revealed the presence and the correct orientation
of several important markers of leukocytes: CD45, CD47 and MHC-I were identified as
key players in determining LLVs biocompatibility, while Leukocyte Associated Function-1
(LFA-1) and Mac-1 contributed to the LLVs targeting ability and bioactivity towards
inflamed endothelium [3]. Recent investigation showed that the coating induced the
formation of a singular protein corona (i.e. the protein adsorption layer) on the
surface of the nanoparticles compared to negative control following in vivo injection.
In addition, the proteolipid coating favored active extravasation of the LLVs in the
tumor vasculature by molecular mechanisms similar to those used by tumor infiltrating
leukocytes. This work shows that is possible to transfer biologically active leukocyte
membrane proteins onto synthetic nanoparticles, thus creating biomimetic carriers
retaining cell-like functions that are not affected by the protein corona effect that
occurs in vivo. The targeting of the inflamed endothelium can be applied to a broad
range of diseases and the approach used to formulate the system could open new avenues
for the fabrication of the next generation of personalized treatments by using as
cell membrane source the immune cells of patients.
References:
[1] Alessandro Parodi, Claudia Corbo, Armando Cevenini, Roberto Molinaro, Roberto
Palomba, Laura Pandolfi, Marco Agostini, Francesco Salvatore, Ennio Tasciotti. Enabling
cytoplasmic delivery and organelle targeting by surface modification of nanocarriers.
Nanomedicine UK. Accepted.
[2] Parodi A. et al. Synthetic nanoparticles functionalized with biomimetic leukocyte
membranes possess cell-like functions. Nature Nanotechnoly. 2013. 8(1): 61-8.
[3] Corbo C, Parodi A, Evangelopoulos M, Engler DA, Matsunami RK, Engler AC, Molinaro
R, Scaria S, Salvatore F, Tasciotti E. Proteomic profiling of a biomimetic drug delivery
platform. Curr Drug Targets.
PJ-003
Visualising gene regulation: a combined proteomic and genomic approach for the structural
analysis of steroid hormone receptor complexes
Andrew Holding1
1Cancer Research UK Cambridge Institute, University of Cambridge
I will present my work on the development of a novel technique for identifying key
protein interactions specific to the growth of tumour cells with the potential to
identify new leads for therapeutic targets. Steroid hormone receptors are intracellular
receptors that initiate signal transduction in response to steroid hormones, including
oestrogen and androgens. Generally, the binding of the steroid to the nuclear receptor
induces the protein to form a dimer and relocate onto the chromatin, although the
order of these events may vary. The location of receptor binding on the chromatin
is defined by specific hormone response elements (HRE). Once located, the receptor
promotes gene activation by the recruitment of other co-factors. It is this process
that makes the complex of receptor protein and co-factors play a pivotal role in the
regulation and activation of genes. The failure to regulate this process correctly
is a key step in the development of several endocrine-driven cancers. For example:
oestrogen receptor positive (ER+) breast cancer is one of the most common forms of
cancer and accounts for 70% of all breast cancer cases. In ER+ tumours, the oestrogen
receptor (ER) drives the tumour growth and cell proliferation. Understanding the interactions
of the ER with other proteins, either directly or indirectly, can provide vital insight
to the regulation of the system that drives this cancer. The progesterone receptor
(PR) has also been implicated in breast cancer, and the androgen receptor (AR) is
a known driver in the majority of prostate cancers. To meet the challenges of elucidating
these systems, we have developed methods to purify and analyse cross-linked regulatory
complexes bound to DNA by mass spectrometry (ChIP-MS). This allows for the enrichment
of proteins involved in gene regulation. ChIP-MS, combined with tandem mass tags (TMT),
makes it possible to realise a quantitative method to investigate the dynamic network
of interactions between proteins within complexes that undertake the regulation of
biological systems. ChIP-seq is a well-established method for identifying where these
protein complexes are bound to the genome. This work focuses on how to combine these
technologies with my previous development of cross-linking coupled mass spectrometry
techniques (XCMS) to provide a strategy for visualising the dynamic organisation of
the proteins on the chromatin.
PJ-004
Global kinetic analysis of caspase protein substrates in cell lysate reveals selective
roles and target specificity
Olivier Julien1, Min Zhuang1, Arun Wiita1, James Wells1
1University of California
Caspases are cysteine proteases that play important roles in development, cell differentiation
and cell death. However, the limited number of known caspase substrates hinders our
understanding of caspase function. Here we performed a non-biased identification and
kinetic analysis of caspase-2 and caspase-6 proteolytic substrates in cell lysate,
using an enzymatic N-termini enrichment approach followed by mass spectrometry. We
identified 235 and 871 potential substrates for the initiator caspase-2 and putative
executioner caspase-6, respectively. Our results not only confirm known substrates
but also identify many more new substrates with the precise location of proteolysis.
Given the emerging roles of caspases-2 and −6 in inflammation and neurodegeneration,
these new substrates may provide molecular insight into the progression of related
diseases. The sequence consensus logo of caspase-2 targets was very similar to a classical
executioner caspase motif (DEVD), while caspase-6 revealed a VEVD motif. Using selected
reaction monitoring (SRM), we quantified the kinetics of proteolysis of a large subset
of these substrates by measuring the appearance of the caspase cleavage product over
time. In the end, we measured 50 and 276 kcat/Km values for individual substrates
cut by caspase-2 and caspase-6, respectively. By comparing these data with our previous
analysis of caspase-3, −7, and −8, we found that substrates that are shared between
caspases are often cleaved at rates that differ by orders of magnitude. Thus, despite
having nearly identical primary sequence motifs, the caspases exhibit remarkable substrate
specificity that may reflect their specialized roles within the cell.
PJ-006
Interactomic and Enzymatic Analyses of Distinct Affinity Isolated Human Retrotransposon
Intermediates
John LaCava1,2, Kelly Molloy1, Martin Taylor3, David Fenyö2, Lixin Dai3, Brian Chait1,
Jef Boeke2, Michael Rout1
1The Rockefeller University, 2New York University School of Medicine, 3Johns Hopkins
University School of Medicine
LINE-1 (L1) retrotransposons are catalysts of evolution and disease whose sequences
comprise a significant proportion of the human genome. Despite tremendous influence
on genome composition, L1 RNAs only encode two proteins. Consequently, L1 particles
include a combination of permissive host factors that are essential to their lifecycle
as well as repressive factors that constitute defenses against L1’s mutagenic activity.
We previously characterized host proteins associated with synthetic and natural human
L1 retrotransposons, as expressed in cell culture, using a combination of techniques
including metabolic labeling and affinity proteomics. To build on these analyses,
we have implemented a series of 2D separations and post-purification treatments to
produce a multi-dimensional interactomic characterization of affinity isolated L1s.
These studies have revealed the presence of at least two populations of putative transposition
intermediates that may exhibit distinctive intracellular localizations. We report
a comprehensive, quantitative survey of the proteins partitioning within these distinct
L1 populations and their associated in vitro activity. Our observations provide a
basis for the classification of L1 interactors with respect to their physical and
functional links, facilitating hypotheses to direct in vivo experimentation.
PJ-007
Polyubiquitin recognition by continuous ubiquitin binding domains of Rad18 probed
by modeling, small-angle X-ray scattering and mutagenesis
Sangho Lee1, Trung Thanh Thach1, Namsoo Lee1, Donghyuk Shin1, Seungsu Han1, Gyuhee
Kim1, Hongtae Kim1
1Department of Biological Sciences, Sungkyunkwan University
Rad18 is a key protein in double-strand break DNA damage response (DDR) pathways by
recognizing K63-linked polyubiquitylated chromatin proteins through its bipartite
ubiquitin binding domains UBZ and LRM with extra residues in between. Rad18 binds
K63-linked polyubiquitin chains as well as K48-linked ones and mono-ubiquitin. However,
the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear.
Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear
polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240)
binds linear polyubiquitin chains (Ub2, Ub3, Ub4) with similar affinity to a K63-linked
one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at
the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed ‘extended
LR motif’ (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by
small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear
Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin
by contacting the proximal ubiquitin moiety. Consistent with the structural analysis,
mutational studies showed that residues in ELRM affect binding with linear Ub2, not
monoubiquitin. In cell data support that ELRM is crucial in Rad18 localization to
DNA damage sites. Specifically E227 seems to be the most critical in polyubiquitin
binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding
domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative
basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that
Rad18(201-240) forms continuous ubiquitin binding domains, comprising UBZ and ELRM,
and provides a structural framework for polyubiquitin recognition by Rad18 in the
DDR pathway at a molecular level.
PJ-008
Optimization of a protein extraction method for the proteomic study of pozol
Cynthia Teresa Leyva-Arguelles1, Carmen Wacher2, Rosario Vera3, Romina Rodríguez-Sanoja1
1Instituto de Investigaciones Biomédicas, UNAM., 2Facultad de Química, UNAM., 3Instituto
de Biotecnología, UNAM
Key words: Proteomics, fermentation, pozol
Pozol is a Mexican traditional no alcoholic beverage elaborated by various ethnic
groups in the southeastern of Mexico. Pozol is obtained from the natural fermentation
of nixtamal (heat- and alkali-treated maize) dough. The main carbohydrate in maize
dough is starch (72-73%), because others such as sucrose, glucose and fructose are
mostly lost during nixtamalization; so, the starch remains as the major carbohydrate
available for fermentation [1]. A wide variety of microorganisms have already been
isolated from the fermentation of pozol; these microorganisms include fungi, yeasts,
lactic acid bacteria, and non-lactic acid bacteria [2]. However, only few bacteria
are amylolytic in this fermentation and all of them are weakly amylolytic [1]. In
an attempt to explain how a very low content of soluble sugars can support a diverse
and abundant microbiota, a proteomic approach was designed to understand the fermentation
of pozol [3]. Nevertheless, the extraction of proteins from pozol remains a limiting
step in proteomic analysis mainly due to the complexity of the sample. On the basis
of the aforementioned reasons, the aim of this work was to obtain a suitable extraction
method of proteins for proteomic analysis. Therefore, the fermentation of pozol was
continued for 48 h and samples were taken at 0, 9, 24 and 48 h. For each sample, the
total sugar content was determined by the Dubois et al. method [4] and protein extraction
was performed by two methods: A) Direct extraction from the dough [3] and B) Initial
extraction of microorganisms and soluble proteins (this work). Comparison between
the two protein methods was performed on two-dimensional gels with silver stain. Then,
gels underwent to image analysis by the image master 2D Platinum software. Comparing
the 2D-gels, more proteins spots were obtained with method B than that with method
A, indicating a more efficient protein extraction with method B. Although, using method
A higher concentration of total proteins was observed, they were mostly maize proteins,
that in turn overlap and reduce the efficiently extraction of the microbial low abundant
proteins. Then, method B allows a better extraction of those low abundant proteins
and removes sample components that may interfere with the determination. These results
could help us to find the proteins involved in carbohydrate metabolism of the microbiota
and finally elucidate the dynamics of pozol fermentation.
Acknowledgements:
Cynthia Leyva-Argüelles is supported by a personal grant from CONACyT, Mexico. This
work is supported by CONACYT grant 131’06:15 and PAPIIT grant IN218714.
References:
[1] Díaz-Ruiz, G. et al. (2003) Appl. Environ. Microbiol. 8: 4367-4374.
[2] Ampe, F. et al. (1999). Appl. Environ. Microbiol. 12: 5464-5473.
[3] Cárdenas, C. et al. (2014) J. Proteomics. 111: 139-147.
[4] Dubois, M. et al. (1956) Anal. Chem. 28: 350-356.
PJ-009
Proteomics and enology: wine yeasts study applications
Jaime Moreno García1, Juan Carlos Mauricio1, Juan Moreno2, Anna Lisa Coi3, Marilena
Budroni3, Teresa García Martínez1
1Department of Microbiology, ceiA3, 2Department of Agricultural Chemistry, ceiA3,
3Dipartimento di Agraria
Proteomics has been applied to the enology field for numerous purposes including fermentation
control, improvement of fermentation processes, ensuring wine quality, etc. According
to Rodriguez et al., (2012), the information provided by wine proteomics is not only
useful for these intentions, but also offers excellent prospects for innovation and
diversification of winemaking processes in the near future. In this context, our group
has focused research on the identification of proteins that might be important for
yeast survival under typical wine elaboration conditions (standard fermentation, Sherry
wine biological aging and sparkling wine second fermentation) as well as proteins
that configure the content of metabolites which are ultimately responsible for wine
quality. By using novel proteomic (OFFGEL fractionator and LTQ Orbitrap XL MS) and
metabolomic techniques (SBSE-TD-GC-MS) we have identified a high amount of up-regulated
proteins involved in processes like oxidative stress response (in biological aging)
or protein biosynthesis (in second fermentation) as well as thirty-three proteins
directly involved in the metabolism of glycerol, ethanol and seventeen aroma compounds
excreted by the yeast under biological aging conditions. Further, in order to validate
proteome data; null mutants of genes codifying proteins up-regulated in the biological
aging condition were constructed. Analyses of correlated phenotypes are in progress.
This technique and its combination with Metabolomics within the enology context will
provide enough knowledge to design or choose yeasts or conditions that satisfy wine
production and/or wine characteristics such as color/aroma/texture/flavour profile
demands of wine-makers and consumers.
PJ-010
Additional binding sites for cytochrome c on its redox membrane partners facilitate
its turnover and sliding mechanisms within respiratory supercomplexes
Blas Moreno-Beltrán1, Antonio Díaz-Quintana1, Katiuska González-Arzola1, Alejandra
Guerra-Castellano1, Adrián Velázquez-Campoy0, Miguel A. De la Rosa1, Irene Díaz-Moreno1
1IBVF, cicCartuja, Universidad de Sevilla - CSIC, 2BIFI - IQFR (CSIC), Universidad
de Zaragoza, 3Departamento de Bioquímica y Biología Molecular Celular, Universidad
de Zaragoza, 4ARAID Foundation, Government of Aragon
Gliding mechanisms of cytochrome c (Cc) molecules have been proposed to shuttle electrons
between respiratory complexes III and IV within plant and mammalian mitochondrial
supercomplexes, instead of carrying electrons by random diffusion across the intermembrane
bulk phase [1-2]. In this work, the binding molecular mechanisms of the plant and
human Cc with mitochondrial complexes III and IV have been analyzed by Nuclear Magnetic
Resonance and Isothermal Titration Calorimetry. Our data reveal that both Cc-involving
adducts possess a 2:1 stoichiometry – that is, two Cc molecules per adduct –. The
presence of extra binding sites for Cc at the surfaces of complexes III and IV opens
new perspectives on the mitochondrial electron transport chain, where membrane respiratory
complexes can be either in independent, free diffusional motion or forming macromolecular
assemblies. In the latter context, such new binding sites for Cc facilitate the turnover
and sliding mechanisms of Cc molecules within supercomplexes. Indeed, the accommodation
of several Cc molecules between complexes III and IV in supercomplexes provide a path
for Cc diffusion from complex III to IV. Such path could have physiological significance
in the electron flow, which is controlled in supercomplexes to optimize the use of
available substrates [3-5].
[1] Genova, G, Lenaz, A. (2013) A critical appraisal of the role of respiratory supercomplexes
in mitochondria. Biol. Chem. 394, 631-639.
[2] De March, M, Demitri, N, De Zorzi, R, Casini, A, Gabbiani, C, Guerri, A, Messori,
L and Geremia, S. (2014) Nitrate as a probe of cytochrome c surface crystallographic
identification of crucial “hot spots” for protein-protein recognition. J. Inorg. Biochem.
135, 58-67.
[3] Moreno-Beltrán, B, Díaz-Quintana, A, González-Arzola, K, Velázquez-Campoy, A,
De la Rosa, MA and Díaz-Moreno, I. (2014) Cytochrome c1 exhibits two binding sites
for cytochrome c in plants. Biochim. Biophys. Acta – Bioenergetics 1837, 1717-1729.
[4] Moreno-Beltrán, B, Díaz-Moreno, I, González-Arzola, K, Guerra-Castellano, A, Velázquez-Campoy,
A, De la Rosa, MA and Díaz-Quintana, A. (2015) Respiratory complexes III and IV can
each bind two molecules of cytochrome c at low ionic strength. FEBS Lett. 589, 476-483.
[5] Moreno-Beltrán, B*, González-Arzola, K*, Martínez-Fábregas, J, Díaz-Moreno, I
and De la Rosa, MA. (2015) Cytochrome c-based signalosome. In Redox proteins in supercomplexes
and signalosomes, Editors: R.O. Louro and I. Díaz-Moreno. Taylor and Francis
Editiorial Group. ISBN: 978-1-4822-5110-4.
PJ-011
Can Bio-functionalities be deciphered from protein sequence information using computational
approaches?
Norbert Nwankwo1
1University of Port Harcourt
Background: The processes of uncovering bio-functionalities such as pharmacological
activities, disease processes, physiological and structural properties by means of
clinical approaches are irrational. This is because they are resource and time consuming.
Sometimes, they involve sophisticated and expensive equipments, reagents and animal
tissues. Contrarily, sequence information-based computerized approaches are rational
and have become relevant in assessing bio-functionalities. They include geno2pheno
[CORECEPTOR] [1], Position-Specific Scoring Matrix (PSSMSI/NSI and PSSMCXCR4/CCR5)
[2], and Informational Spectrum Method (ISM)-based phylogenetic analysis (ISTREE)
[3]. Aim: This presentation demonstrates how bio-functionalities could be deciphered
from sequence information using computational approaches. Method: ISM procedure and
peptides, VIPMFSALS and CAPAGFAIL are engaged. Results: Protein sequences of the peptides
are converted into bio-functionality (Affinity). Affinity between the two peptides
is demonstrated as significant amplitudes at the point of common interaction also
referred to as Consensus Frequency, signifying remarkable affinity. Discussions: Bio-functionalities
of bio-molecules are known to be expressed in one or two genes, which have been found
to provide as much biological information as the bio-molecules. This indicates that
biological characteristics, represented in these genes and proteins can now be extracted
from their sequence information. For example, multi-drug resistances arising from
a variety anti-microbial agent from several classes including alkaloids, flavonoids,
etc can be retrieved from the sequence information of their encoding genes (MDR1 and
MDR11). Similarly, translation of HIV infection to AIDS disease can be extracted from
the protein sequence alterations in the HIV gp120. Similarly, effectiveness of anti-retroviral
agent, Maraviroc on the HIV isolate H2BX2 and NDK can be deciphered from the sequence
information of their V3 domain using geno2pheno [CORECEPTOR] [1]. Sequence information-based
deciphering of bio-functionalities using ISM-based techniques has fetched calculation
of biological functionalities, designing of biomedical device called Computer-Aided
Drug Resistance Calculator, the understanding of the mechanism of HIV progression
to AIDS [4], and others. They have compared the efficacies of drugs and vaccines,
which formed the basis for the Innocentive Award (ID 9933477) for Assessing Vaccine
Potency. Conclusions: Deciphering biological features without engaging reagents, equipments
and animal tissues but biological data such as sequence information is one novel,
feasible, rational and computerized research accomplishment that will revolutionize
translation of therapeutic candidates into therapies.
References:
1. Obermeier M, Ehret R, Berg T, et al, “Genotypic HIV-coreceptor tropism prediction
with geno2pheno [CORECEPTOR]: differences depending on HIV-1 subtype”, Journal of
the International AIDS Society, vol. 15(Suppl 4), pp. 1821-1824, 2012.
2. Jensen MA, Coetzer M, van ’t Wout AB, et al, “A reliable phenotype predictor for
human immunodeficiency virus type 1 subtype C based on Envelope V3 sequences”, Journal
of Virology, vol. 80, pp. 4698-4704, 2006.
3. Open Web Server for Informational Spectrum-based Phylogenetic Analysis (ISTREE).
Available: http://istree.bioprotection.org/. Accessed 2012 Nov 15.
4. Nwankwo N. 2012. Signal processing-based Bioinformatics methods for characterization
and identification of Bio-functionalities of proteins. PhD Thesis (submitted). De
Montfort University, Leicester, United Kingdom; available at www.openthesis.org
PJ-012
Prediction of cleavage specificity in HCV NS3/4A serine protease and AdV2 cysteine
protease systems by biased sequence search threading
Gonca Ozdemir Isik1, A.Nevra Ozer1
1Department of Bioengineering,Faculty of Engineering,Marmara University
Proteases are enzymes which recognize specific substrate sequences and catalyze the
hydrolysis of designated peptide bonds to activate or degrade them. Due to the biological
importance of proteases, it is particularly important to identify the recognition
and binding mechanisms of protease-substrate complex structures in drug development
studies. The assessment of substrate specificity in protease systems is crucial, where
interpreting the adaptability of substrate residue positions can be useful in understanding
how inhibitors might best fit within the substrate binding sites and aid in the design
of potent selective inhibitors. Substrate specificity is generally determined by the
amino acid profile, structural features and distinct molecular interactions. Besides
experimental methods, computational tools for prediction of natural substrate cleavage
sites, such as threading, have emerged as useful alternative approaches to provide
valuable insights into complex enzyme-substrate interactions. In this work, the substrate
variability and substrate specificity of the Hepatitis C virus (HCV) NS3/4A serine
protease and the Adenovirus 2 (AdV2) cysteine protease was investigated by the biased
sequence search threading (BSST) methodology. Using available crystal structures of
the proteases, the template structures for the substrate-bound proteases were created
in silico by performing various peptide building and docking procedures followed by
energy minimization and molecular dynamics (MD) simulations. BSST was performed starting
with known binding, nonbinding and some random peptide sequences that were threaded
onto the template complex structures, and low energy sequences were searched using
low-resolution knowledge-based potentials. Then, target sequences of yet unidentified
potential substrates were predicted by statistical probability approaches applied
on the low energy sequences generated. The results show that the majority of the predicted
substrate positions correspond to the natural substrate sequences with conserved amino
acid preferences, while some positions exhibit variability. For NS3/4A serine protease
cleavage, the significant selection for Pro at P2 and Cys at P1 positions is observed
at the predicted sequences. These positions are important as they surround the cleavage
site in the three-dimensional structure, and are probably less tolerant to change.
Moreover in previous studies, Cys at P1 position has been shown to be the dominant
determinant for cleavage efficiency, while Cys, Pro and Glu at P2 position have also
been shown to be correlated with increased cleavage efficiency of NS3/4A protease.
For AdV2 cysteine protease, on the other hand, BSST produces similar significant results
for both type 1 (XGX-G) and type 2 (XGG-X) consensus cleavage sites, where P2 and
P1’ positions have Gly with highest percentage in type 1 (XGX-G) while P2 and P1 positions
have Gly in type 2 (XGG-X). These indicate that the BSST seems to provide a powerful
methodology for predicting the substrate specificity for the HCV NS3/4A serine protease
and AdV2 cysteine protease, which are targets in drug discovery studies.
PJ-013
Protein plasticity improves protein-protein binding description
Chiara Pallara1, Juan Fernández-Recio1
1Joint BSC-CRG-IRB Research Program In Computational Biology
An accurate description of protein-protein interactions at atomic level is fundamental
to understand cellular processes. However the current structural coverage of protein-protein
interactions (i.e. available experimental structures plus potential models based on
homologous complex structures) is below 4% of the estimated number of possible complexes
formed between human proteins.1,2 For these reasons, computational docking methods
aim to become a complementary approach not only to solve the structural interactome
but also to elucidate the basis of the protein-protein association mechanism. In spite
of the advances in protein-protein binding description by docking, dealing with molecular
flexibility is a major bottle-neck, as shown by the recent outcomes of the CAPRI (Critical
Assessment of PRediction of Interactions) experiment.3 This data clearly confirms
that the protein dynamics plays a key role in protein-protein association. The use
of conformational ensembles generated from unbound protein structures in combination
with computational docking simulations might represent a more realistic description
of protein-protein association. Here, we present the first systematic study about
the use of precomputed unbound ensembles in docking, as performed on a set of 124
cases of the Protein-Protein Docking Benchmark 3.0.4 The primary aim of our work is
to understand the role of the protein conformational heterogeneity in protein-protein
recognition. To do this, small conformational ensembles were automatically generated
starting from the unbound docking partners, and then an extensive analysis of their
binding properties was performed in the context of pyDock docking scheme.5 The results
show that considering conformational heterogeneity of interacting proteins can improve
docking description in cases that involve intermediate conformational changes in the
unbound-to-bound transition. More interestingly, we found that protein plasticity
increases chances of finding conformations with better binding energy, not necessarily
related to bound geometries. The relevance for future docking methodology development
and for understanding protein association mechanism will be discussed.
References:
1. Venkatesan K, Rual JF, Vazquez A, Stelzl U, Lemmens I, Hirozane-Kishikawa T, Hao
T, Zenkner M, Xin X, Goh KI, Yildirim MA, Simonis N, Heinzmann K, Gebreab F, Sahalie
JM, Cevik S, Simon C, de Smet AS, Dann E, Smolyar A, Vinayagam A, Yu H, Szeto D, Borick
H, Dricot A, Klitgord N, Murray RR, Lin C, Lalowski M, Timm J, Rau K, Boone C, Braun
P, Cusick ME, Roth FP, Hill DE, Tavernier J, Wanker EE, Barabasi AL, Vidal M. An empirical
framework for binary interactome mapping. Nat Methods 2009;6:83-90.
2. Stumpf MP, Thorne T, de Silva E, Stewart R, An HJ, Lappe M, Wiuf C. Estimating
the size of the human interactome. Proc Natl Acad Sci U S A 2008;105:6959-64.
3. Bonvin A. Coming to peace with protein complexes? 5th CAPRI evaluation meeting,
April 17-19th 2013–Utrecht. Proteins, 2013; 81, 12, 2073-4.
4. Hwang H, Pierce B, Mintseris J, Janin J, Weng Z. Protein-protein docking benchmark
version 3.0. Proteins, 2008; 73, 705-9.
5. Cheng TM, Blundell TL, Fernandez-Recio J. pyDock: electrostatics and desolvation
for effective scoring of rigid-body protein-protein docking. Proteins 2007;68:503-15.
PJ-014
Affimers, new affinity reagents for life science research
Vincent Puard1, Kit-Yee Tan1, Kurt Baldwin1, Enitan Carrol2, Rebecca Patisson3, Rob
Beynon3, Christian Tiede4, Michael McPherson4, Darren Tomlinson4, Paul Ko Ferrigno1
1Avacta Life Sciences, 2Institute of Infection and Global Health, University of Liverpool,
3Centre for Proteome Research, University of Liverpool, 4Biomedical Health Research
Centre, University of Leeds
Purpose of the research: There is increasing interest in the development of protein
scaffolds that can be used to develop affinity reagents that are alternatives to antibodies.
The Affimer scaffold is based on the cystatin protein fold. The Affimer scaffold is
biologically inert, biophysically stable and capable of presenting a range of designed
or random binding surfaces defined by peptides inserted at 2 different loops. The
result is highly specific, high affinity interactions with a wide range of targets
including ones that are inaccessible to antibodies. Affimers are designed to work
in the same way as the very best antibodies, but with a number of key advantages.
Affimers are quick to develop (typically 7 weeks) without using animals. They contain
no disulphide bonds, are expressed easily in E. coli and have no batch to batch variability.
Affimers are small molecules (108 aa, ∼12 kDa), robust and stable (resistant to pH
range, thermally stable and not sensitive to EDTA). Affimers can be a direct replacement
for antibodies – no process or workflow change required – and perform identically
to antibodies in assays such as ELISA, FACS, IHC, western blots, affinity purification,
microarray and potentially therapeutics. We describe some applications of the technology
in regards of Affimer development for custom targets on one hand and for the biomarker
discovery workflow using Affimer microarrays on the other. Main results: By screening
of our very large (3 x 1010) library against Yeast SUMO protein we identified Affimers
with high affinity allowing their use for ELISA. Moreover, no cross-reactivity was
observed when Affimers were used on western blots leading to a unique band specific
to Yeast SUMO when compared to human proteins. A library of 25,000 random Affimers,
expressed in E. coli, was printed on glass microscope slides and challenged with plasma
from children (n=104) with sepsis and from healthy children (n=24). Unsupervised hierarchical
clustering based on the 25,000 Affimers allowed differentiation between the control
and patient samples. 200 Affimers were found to differentially bind proteins between
the 2 groups with a > 2 fold change. The Affimer arrays identified a strong signature
of sepsis and ROC curve analysis allowed confident prediction of disease (AUROC of
0.9). Affinity purification and preliminary mass spectrometry analysis identified
known biomarkers of sepsis and also potentially novel biomarkers not previously associated
with this disease. Major conclusions: This work demonstrates the scope of Affimer
affinity reagents to develop alternative binders to antibodies, where Affimers perform
identically in most assays without the disadvantages associated with antibodies. Moreover,
Affimers enable a new protein microarray-based biomarker-discovery workflow and we
predict that array-based validation of signatures identified using Discovery Arrays
prior to affinity purification and mass spectrometry will offer a cost- and time-effective
methodology compared to purely mass spec-driven workflows.
PJ-015
NMR study of ERK-mediated hyperphosphorylation of the neuronal Tau protein
Haoling Qi1, François-Xavier Cantrelle2, Amina Kamah1, Clément Despres1, Sudhakaran
Prabakaran2, Jeremy Gunawardena2, Guy Lippens1, Isabelle Landrieu1
1UMR 8576 CNRS-USTL, Lille University, 2Department of Systems Biology, Harvard Medical
School
Tau pathologies, called ‘tauopathies’, are related to several neurodegenerative diseases
including Alzheimer Disease (AD). In AD, Tau protein is observed hyper-phosphorylated
and aggregated as Paired Helical Filament (PHF). The neuronal Tau protein is an Intrinsically
Disordered Proteins (IDPs). Nuclear Magnetic Resonance spectroscopy (NMR) is here
used to study the Tau protein phosphorylations and Protein-Protein Interactions (PPIs).
In in vitro assays, Tau phosphorylation by rat brain extract is considered as an hyperphosphorylation
model that was furthermore pointed out to enable Tau aggregation [1]. In a first step,
we have identified all the phosphorylation sites of rat brain extract phosphorylated-Tau,
using the analytical capacity of NMR. We showed that the protein is modified at 20
Ser/Thr sites. Among the kinases that we have characterized so far using Tau as substrate,
only the extracellular signal-regulated kinase2 (ERK2) shows an ability to modify
in vitro Tau protein on so many sites. We have indeed identified 14 phosphorylated
Ser/Thr-Pro motifs out of 18 potential phosphorylation sites in the sequence of full
length 441-residue Tau. In addition, we showed using Transmission Electron Microscope
(TEM) a similar in vitro aggregation capacity of ERK-phosphorylated Tau protein compared
to that of rat brain extract phosphorylated-Tau. This shows that phosphorylation by
the ERK kinase generates an hyperphosphorylated Tau. Given the high efficiency of
ERK towards Tau, we have next looked into the mechanism of Tau recognition. ERK kinase
possesses two well-characterized docking domains: D Recruitment Sites (DRS) and F
Recruitment Sites (FRS), which recruit complementary docking sites and increase the
specificity and efficiency of the interaction with both its upstream regulators and
downstream substrates [3]. As the interaction between Tau protein and ERK2 kinase
is analyzed by NMR spectroscopy, multiple sites of interaction are observed along
the Tau sequence, similar to DRS docking sites, all located in the so-called microtubule
binding domain of Tau. These sites are short sequences loosely matching the reported
consensus for D sites ψ1-3φxφ (ψ, φ, and x refer to positively charged, hydrophobic,
or any intervening residues, respectively) [3], and also the reverse sequence φxφψ
1-3.To confirm the mapping of the interaction, two Tau recognition sites were produced
as recombinant peptides of about 20 amino-acid in fusion with an N-terminal His-tag
Sumo. Interaction assays using 2D [1H, 15N] HSQC spectra of the peptides confirm their
binding to ERK kinase. The potential of these peptides to inhibit ERK activity with
Tau as substrate is now being investigated.
[1]: A.C.Alonso, T.Zaidi. et al. PNAS. 2001, 98: 6923-6928
[2]: P.D. Mace, Y. Wallez, M.F.Egger. et al. Nature Communications. 2013, 4: 1681
[3]: A. Garai, A. Zeke, G. Gogl. et al. Sci. Signal. 2012, 5, ra74
PJ-016
How binding incorrect partners can lead to the prediction of correct interfaces: Results
from a massive cross-docking study on proteins.
Sophie Sacquin-Mora1, Lydie Vamparys1, Alessandra Carbone2
1Laboratoire de Biochimie Théorique, CNRS UPR9080, 2Génomique Analytique, Université
Pierre et Marie Curie, CNRS UMR7238
While rigid-body docking has become quite successful for predicting the correct conformations
of binary protein complexes, determining whether two given proteins interact remains
a difficult problem. Successful docking procedures often give equally good scores
for pairs of proteins for which there is no evidence of interaction. Studies investigating
what we define as the ’pre-docking’ problem via in silico approaches have only recently
become feasible with the help of supercomputers and grid-computing systems. In a previous
work, on a restricted set of protein complexes, we showed how predictions of interacting
partners could be greatly improved if the location of the correct binding interface
on each protein was known. Experimentally identified complexes are found to be much
more likely to bring these two interfaces into contact, at the same time as yielding
good interaction energies. We present data from a complete cross-docking (CC-D) study
of a database of 168 proteins, including the treatment of more than 14,000 potential
binary interactions. The performance of the interaction index we developed to predict
binding probability compares well with other methods. By studying the interaction
of all potential protein pairs within a dataset, CC-D calculations can also help to
identify correct protein interaction interfaces. The present large-scale study also
reveals the influence of various protein families (enzyme-inhibitor, antibody-antigen,
antigen-bound antibody, etc.) on binding specificity, showing, in particular, the
distinctive behavior of antigenic interfaces compared to enzymes, inhibitors or antibodies.
The performance of our approach is encouraging. Although identifying interaction interfaces
significantly helps in the identification of interacting proteins, further refinements
will be necessary to make in silico cross-docking a viable alternative to high-throughput
experimental methods.
PJ-017
Whole-protein mass spectrometry reveals global changes to histone modification patterns
in hypoxia
Sarah Wilkins1, Kuo-Feng Hsu1, Christopher Schofield1
1Chemistry Research Laboratory, Oxford University
Cells respond to limiting oxygen availability (hypoxia) by altering the gene expression
profile. This primarily involves changes at the level of transcription via the activity
of hypoxia-responsive transcription factors, although increasing evidence suggests
that changes in chromatin structure (i.e. from a condensed ‘silent’ state to a more
open or ‘active’ state) are required in order for transcription to take place. In
particular, post-translational modifications (PTMs) to histones have an important
regulatory function in gene expression under hypoxic conditions. The N-terminal tails
of histone proteins are accessible to a set of enzymes capable of ‘writing’ and ‘erasing’
PTMs including acetylation, methylation, ubiquitylation, SUMOylation and phosphorylation.
To date, studies in hypoxia have employed antibody-based methods to investigate changes
in histone modifications, and so have focused on individual marks in isolation. The
interplay between coexisting PTMs is thought to be much more important than the effect
of any single mark. Therefore, a global view of the histone modification profile is
essential to gain a complete understanding of the function of histone PTMs and their
roles in gene regulation. In this study, we apply whole protein mass spectrometry
to investigate hypoxia-induced changes in histone marks. This ‘top-down’ approach
provides insight into combinational modification patterns that are difficult to establish
by antibody-based methods or peptide MS analysis. We investigated changes in the global
PTM profiles of histones from a range of human cell-lines and tissues under severe
hypoxia (<0.1% O2). We find that hypoxia causes a shift in the overall profile towards
a more highly modified state, with significant changes in methylation and phosphorylation.
Marked changes in histone PTMs were also observed following treatment of cells with
epigenetic inhibitors and commonly used hypoxia mimetics, including several iron chelators
currently in clinical trials for the treatment of anaemia. Finally, we show that this
method can be used to identify the histone variant H2AX, whose phosphorylation at
serine 139 is an indicator of double-stranded DNA breaks in cancer. Overall, these
data provide important insights into the epigenetic changes associated with hypoxia
in normal and disease contexts. We hope to further develop this method in combination
with different labelling strategies to enable quantitative analysis of histonemodifications
in cells.
PJ-018
Mass spectrometry-based protein biomarker discovery in neurodevelopmental disorders
Kelly Wormwood1, Armand Ngounou Wetie1, Laci Charette2, Jeanne Ryan2, Emmalyn Dupree1,
Alisa Woods0, Costel Darie1
1Clarkson University, 2SUNY Plattsburgh
Neurodevelopmental disorders are a group of disorders in which the development of
the central nervous system is disturbed. These are very common with approximately
15% of children in the United States ages 3 to 17 being affected by at least one disorder.
Examples include Autism Spectrum Disorder (ASD) and Smith-Lemli-Opitz Syndrome (SLOS).
ASD affects approximately 1/63 children in the United States and is characterized
by repetitive behaviors, communication deficits and impairments in social interactions.
There is currently no biological diagnosis or known cause of ASD. SLOS is characterized
by a cholesterol deficiency due to a mutation on the 7DHCR gene. Approximately 1/20,000
babies are born with SLOS. Diagnosis is achieved by measuring cholesterol and 7-dehydrocholesterol
(7DHC) levels in the blood, however, there is currently no proven treatment for SLOS.
Because of this, research is increasing to determine biomarkers for these disorders.
Here, samples from people with ASD (sera and saliva) and SLOS (saliva), and matched
controls were analyzed using a combination of gel electrophoresis (Tricine-PAGE, SDS-PAGE
and Blue Native PAGE), in gel digestion or insolution digestion and nanoliquid chromatography-tandem
mass spectrometry (nanolC-MS/MS) to investigate differences between the proteomes
of people with these neurodevelopmental disorders and matched controls. Several alterations
in protein expression were identified. These differences may lead to potential biomarkers
for diagnosis, possible therapeutic targets and an altogether better understanding
of the disorders.
PJ-019
Understanding protein recognition using structural features
Manuel A. Marin-Lopez1, Joan Planas-Iglesias2, Jaume Bonet3, Daniel Poglayen1, Javier
García-García1, Narcís Fernández-Fuentes1, Baldo Oliva1
1Structural Bioinformatics Lab (GRIB-IMIM), Department of Experimental and Health,
Universitat Pompeu Fabra, 2Division of Metabolic and Vascular Health, University of
Warwick, 3Laboratory of Protein Design & Immunoengineering, School of Engineering,
Ecole Polytechnique Federale De Lausanne
Protein-Protein interactions (PPIs) play a crucial role in virtually all cell processes.
Thus, understanding the molecular mechanism of protein recognition is a critical challenge
in molecular biology. Previous works in this field show that not only the binding
region but also the rest of the protein is involved in the interaction, suggesting
a funnel-like recognition model as responsible of facilitating the interacting process.
Further more, we have previously shown that three-dimensional local structural features
(groups of protein loops) define characteristic patterns (interaction signatures)
that can be used to predict whether two proteins will interact or not. A notable trait
of this prediction system is that interaction signatures can be denoted as favouring
or disfavouring depending on their role on the promotion of the molecular binding.
Here, we use such features in order to determine differences between the binding interface
and the rest of the protein surface in known PPIs. Particularly, we study computationally
three different groups of protein-protein interfaces: i) native interfaces (the actual
binding patches of the interacting pairs), ii) partial interfaces (the docking between
a binding patch and a non-interacting patch), and iii) back-to-back interfaces (the
docking between non-interacting patches for both of the interacting proteins). Our
results show that the interaction signatures in partial interfaces are much less favoured
than the ones observed in native and back-to-back interfaces. We hypothesise that
this phenomenon is related to the dynamics of the molecular association process. Back-to-back
interfaces preserve the exposure of the real interacting patches (thus, allowing the
formation of a native interface), while in a partial interface one interacting patch
is sequestered and becomes unavailable to form a native interaction.
PJ-020
Structural characterization of the cytoplasmic mRNA export platform
Javier Fernandez-Martinez1, Yi Shi2, Seung Joong Kim3, Paula Upla4, Riccardo Pellarin3,
Daniel Zenklusen5, David L. Stokes4, Andrej Sali3, Brian T. Chait2, Michael P. Rout1
1Laboratory of Cellular and Structural Biology, The Rockefeller University., 2Laboratory
of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller Univ., 3University
of California, San Francisco, 4The New York Structural Biology Center, 5Department
of Biochemistry, Faculty of Medicine, University of Montreal
mRNA biogenesis is an intricate process that begins within the nucleus and culminates
with the remodeling and nuclear export of the mRNP particles through the nuclear pore
complex (NPC). Defects in this conserved mechanism have been shown to cause serious
human diseases. The protein assembly that performs the last steps in mRNP biogenesis
and export is located at the cytoplasmic face of the NPC and is formed by 14 different
proteins, organized into several subcomplexes whose arrangement and molecular architecture
are poorly understood. In this study we applied an integrative approach, combining
cross-linking and mass spectrometry (CX-MS), electron microscopy and available high-resolution
structures, to describe the molecular architecture of the endogenous NPC cytoplasmic
mRNP export machinery. We generate a hybrid, close-to-atomic structure of the yeast
native Nup82 complex, the core of the assembly. Our map also reveals how the Nup82
complex organizes the entire cytoplasmic mRNP export machinery, and how this in turn
docks into the architectural core of the NPC. Mapping of phenotypic profiles into
our structures allows us to generate a first functional map of the ensemble. We expect
that our map will serve as a framework to understand the molecular mechanisms underlying
this key step of mRNP biogenesis.
PJ-021
Study of candidate proteins to pore associated with P2X7 receptor in different cell
types
Carla Oliveira1, Anael Alberto1, Mônica Freitas2, Luiz Alves1
1Laboratório de Comunicação Celular – FIOCRUZ, 2Centro Nacional de Ressonância Magnética
Nuclear – UFRJ
Aim: The P2X7R is a purinergic receptor, which differs from others subtypes due to
its structural and pharmacological characteristics. When exposed for extended time
or to high concentrations of its agonist (ATP), promotes an increase in membrane permeability,
allowing the passage of molecules up to 900Da. There is a controversy among several
authors that leave in doubt if this receptor needs a second protein for the pore formation
and which protein could be. We select five pore-forming proteins: TRPV1, TRPA1, Connexins-43
(Cx-43), Pannexin-1 (Panx-1) and VDAC. We believe that different mechanisms and proteins
could be associated with P2X7R, depending on the cell type and their microenvironment
stimuli. In this context, our main goal is identify possible proteins that could be
associated with the P2X7R pore in different cells and species. Methods and Results:
We started with RT-PCR technique of cell lines: J774.G8, N2A, U373, U937, HEK-293
and primary cells from Wistar mouse and Swiss mice. We used different primers and
PCR cycle for each target at different species. We observed that the P2X7, Panx-1
and Cx-43 are the most abundant and are present in all cell types except the absence
of P2X7 in U373 cells and Panx-1 in mice macrophages and U373 cells (n>3). However,
TRPV1 was seen at N2A and U937 cells and TRPA1 in and primary cells from mouse and
mice and in J774.G8 cells (n>3). Regarding to the VDAC, it is present in mouse macrophages,
J774.G8 and HEK-293 cells (n>3). The further steps, we verified if those proteins
could be physically associated with the P2X7R. We co-immunoprecipitated the P2X7R
of J774.G8 (with or without ATP), mice macrophages, HEK-293 and U937 cells. The samples
were applied in two separated 12.5% bis/acrylamide gels: one destined to Mass Spectrometry
(MS) and the other to Western Blot. At this point, we confirmed the presence of P2X7R,
and observed several others proteins associated to P2X7R at different cell conditions,
mainly when we exposed, J774.G8 cell, to 5 mM ATP (n=3). At this condition, we found
by MS, Hsp70, 75, and 90; alpha and β tubulin; myosin Va; alpha, β and γ actin; malate
and lactate dehydrogenase (n=1). Although U977 and HEK-293 had not received ATP treatment,
we found several proteins associated to P2X7. The next step was to immunoprecipitated
those proteins in J774.G8 (treated or not with ATP) and use it to verify if P2X7 are
physically associated to them. As result we saw the P2X7 associated to Panx-1 in J774.G8
cells. Conclusion: We conclude that the P2X7R activated by extracellular ATP triggers
the recruitment of variety different proteins. At this condition, we can suggest that
maybe there is a conformational change, regardless of the numerous recruitment structural
proteins. In addition, apparently, the pore-forming protein Pannexin-1 is associated
with P2X7R, and the others pore forming proteins (VDAC, Cx-43, TRPV1, TRPA1) seems
not be linked to P2X7R at J774.G8 cells
PJ-022
CABS-dock web server for protein-peptide docking with significant conformational changes
and without prior knowledge of the binding site
Mateusz Kurcinski1, Michal Jamroz1, Maciej Blaszczyk1, Andrzej Kolinski1, Sebastian
Kmiecik1
1Department of Chemistry, University of Warsaw
Protein-peptide interactions play a key role in cell functions. Their structural characterization,
although very challenging, is important for discovery of new drugs. Based on our methodology
for highly efficient simulation of proteins [1, 2], we developed the CABS-dock web
server for protein-peptide molecular docking [3]. While other docking algorithms require
pre-defined localization of the binding site, CABS-dock doesn’t require such knowledge.
Given a protein receptor structure and a peptide sequence (and starting from random
conformations and positions of the peptide), CABS-dock performs simulation search
for the binding site allowing for full flexibility of the peptide and small fluctuations
of the receptor backbone [3-5]. This protocol was extensively tested over the largest
dataset of non-redundant protein-peptide interactions available to date (including
bound and unbound docking cases) [3]. For over 80% of the dataset cases, we obtained
models with high or medium accuracy (sufficient for practical applications). CABS-dock
method for coupled binding site search and protein-peptide docking can be easily complemented
by other computational tools (e.g. high-resolution docking refinement protocols) or
experimental data to improve the results of the docking experiment. CABS-dock web
server is freely available at http://biocomp.chem.uw.edu.pl/CABSdock
References:
[1] Jamroz M, Kolinski A, Kmiecik S. (2013) CABS-flex: Server for fast simulation
of protein structure fluctuations. Nucleic Acids Res. 41, W427-31.
[2] Blaszczyk M, Jamroz M, Kmiecik S, Kolinski A. (2013) CABS-fold: Server for the
de novo and consensus-based prediction of protein structure. Nucleic Acids Res. 41,
W406-11.
[3] Kurcinski M, Jamroz M, Blaszczyk M, Kolinski A, Kmiecik S. (2015) CABS-dock web
server for the flexible docking of peptides to proteins without prior knowledge of
the binding site. Nucleic Acids Res. doi: 10.1093/nar/gkv456.
[4] Kurcinski M, Kolinski A, Kmiecik S. (2014) Mechanism of Folding and Binding of
an Intrinsically Disordered Protein As Revealed by ab Initio Simulations. Journal
of Chemical Theory and Computation. 10, 2224-2231.
[5] Blaszczyk M, Kurcinski M, Kouza M, Wieteska L, Debinski A, Michal J, Andrzej K,
Kmiecik S. (2015) Modeling of protein-peptide interactions using the CABS-dock web
server for binding site search and flexible docking. Methods (submitted), preprint
at arXiv:1505.01138.
PJ-023
Web server tools for modeling of protein structure, flexibility, aggregation properties
and protein-peptide interactions
Maciej Blaszczyk1, Michal Jamroz1, Mateusz Kurcinski1, Agata Szczasiuk1, Andrzej Kolinski1,
Sebastian Kmiecik1
1University of Warsaw, Faculty of Chemistry
Recently, we developed a series of molecular modeling tools for structure-based studies
of protein functions and interactions. These tools are publicly available as web servers
that are easily operated even by non-specialists: CABS-fold server for protein structure
prediction [1]; CABS-flex server for modeling of protein structure flexibility [2];
Aggrescan3D server for prediction of protein aggregation propensities and rational
design of protein solubility [3]; and CABS-dock server for prediction of peptide binding
sites and peptide docking [4]. The web servers are freely available from the laboratory
website: http://biocomp.chem.uw.edu.pl/tools
References:
[1] Blaszczyk M, Jamroz M, Kmiecik S, Kolinski A. (2013) CABS-fold: Server for the
de novo and consensus-based prediction of protein structure. Nucleic Acids Res. 41,
W406-11.
[2] Jamroz M, Kolinski A, Kmiecik S. (2013) CABS-flex: Server for fast simulation
of protein structure fluctuations. Nucleic Acids Res. 41, W427-31.
[3] Zambrano R, Jamroz M, Szczasiuk A, Pujols J, Kmiecik S, Ventura S. (2015) AGGRESCAN3D
(A3D): server for prediction of aggregation properties of protein structures. Nucleic
Acids Res. doi: 10.1093/nar/gkv359.
[4] Kurcinski M, Jamroz M, Blaszczyk M, Kolinski A, Kmiecik S. (2015) CABS-dock web
server for the flexible docking of peptides to proteins without prior knowledge of
the binding site. Nucleic Acids Res. doi: 10.1093/nar/gkv456.
PJ-024
Developing a technique to detect deamidated proteins and peptides using Rig-I
Sandy On1, Pinghui Feng2
1University of Southern California, Keck School of Medicine, 2USC Norris Comprehensive
Cancer Center
Developing a Technique to Detect Deamidated Proteins and Peptides Using Rig-I Sandy
On, Pinghui Feng University of Southern California, Norris Comprehensive Cancer Center,
Department of Microbiology, and Molecular Biology, Los Angeles CA Perhaps the most
notable type of post-translational modification of proteins and peptides into a higher
order structure is deamidation of asparagine and glutamine. Deamidation occurs when
an amine group is removed, degrading the molecule for purpose of regulating intracellular
levels. Previous studies have demonstrated that this notable post translational modification
has been uncovered over time for use in DNA recombinant technology as well as use
as a biological clock to facilitate the rapid turnover of biologically important components
of the cell. While the effects of this non-enzymatic chemical reaction have been widely
studied, the method to uncover modification sites over a large quantity of proteins
remains an issue. One of the most common types of deamidation is of asparagine and
glutamine residues. At this time, most researchers will depend on mass spectrometric
based proteomic techniques for identification of these post-translational sites. The
issue is that mass spectral analysis of deamidated proteins and peptides is complication
and can lead to misassigned identification attributed by an overlapping of 13C peak
of the amidated form with the deamidated monoisotopic peak; these two peaks are only
separated by 19.34 mDa. While these issues can be mediated by using a mass spectrometer
with a high mass measurement accuracy, and high resolving power, it is essential to
establish simpler methods for identifying substrates that have undergone deamidation.
If deamidation is present, different protein bands will be exhibited in the western
blot, which will be compared to a triple mutant RIG-I, which resists deamidation,
to observe the location of this modification on the protein. With enough testing,
I will determine specific sites of digestion and use this information to make conclusions
of unknown proteins. I will make results regarding whether the protein has been modified
based on the digestion sites. I will use mass spectrophotometry analysis to compare
the proteins on a wider scale and double check my results. I have narrowed it down
to a couple of different digestion sites that indicate deamidation. Though the analysis
work can be tedious, it is crucial to ensure the sites we isolate are accurate in
order to establish this technique. From my research, we can apply this method for
wider scale use such as in clinical settings. In areas of inflammation of Parkinson’s’
patients, we can review specifically the infected cells versus uninfected and isolate
the proteins, usually deamidated, responsible often smaller in size and more specific.
In addition, research articles have already shown that suppressing modification of
certain cells such as Bcl-xl playing a major in leading the regulation of cancer cell
death by apoptosis. By leading the discovery of a simpler methods to uncovering deamidation
in cells, researchers will more easily and quickly be able to scan through various
proteins, some of which discovered eventually may play pivotal roles in cancer research.
PJ-025
Mass spectrometric evaluation of recombinant hemagglutinin structure conformations
Joanna Szewczak1, Anna Bierczyñska-Krzysik1, Agnieszka Romanik-Chruścielewska1, Iwona
Sokołowska1, Marcin Zieliński1, Piotr Baran1, Violetta Sączyńska1, Małgorzata Kęsik-Brodacka1,
Drota Stadnik1, Grażyna Płucienniczak1
1Institute of Biotechnology and Antibiotics
Influenza virus (IV) hemagglutinin (HA) is a homotrimeric integral membrane glycoprotein
that mediates receptor-binding and membrane fusion. It constitutes the prominent viral
surface antigen and a main target for neutralizing antibodies. Bacterial, recombinant
HA-based vaccines indicate high potential to confer protection against highly pathogenic
(HP) avian IV (AIV) H5N1 and arise as alternative for the traditional egg- or cell
culture-based manufacturing. Relatively short time of bacterial HAs production can
be of great importance in case of a pandemic. Escherichia coli produced protein, based
on the HA sequence of A/swan/Poland/305-135V08/2006(H5N1) HPAIV*, has been successfully
expressed in the form of inclusion bodies at Institute of Biotechnology and Antibiotics.
Refolded and purified antigen was obtained in a soluble form, isolated by reversed
phase HPLC and identified with peptide mass fingerprinting using matrix-assisted laser
desorption/ionization time of flight mass spectrometry (MALDI TOF/TOF MS). The performed
research in a great extent allowed to confirm the amino acid sequence of the recombinant
HA (rHA) assumed based on the cDNA and allowed to establish the location of a total
of six disulfide bridges. However, during purification and storage of the rHA, apart
from desired higher order rosette-like structures of the protein, other non-native
species resulting from posttranslational modifications, misfolding, aggregation and
degradation may occur what results in reduced vaccine potency. Here, besides the properly
folded monomers, we indicate non-native aggregates induced by disulfide crosslinking.
Moreover, several free cysteine residues and unexpected intrachain S-S were identified
in rHA tryptic peptide maps. Cys 43 was found most susceptible to formation of disulfide
bridges between the distinct chains of rHA. The above findings allow to assume that
not all rHA particles fold to form the native structure. Reduced Cys residues exhibit
tendency to undergo oxidation and uncontrolled S-S creation during storage. This may
lead to activity drop and of non-native multimer formation. A covalent modification
of several peptides of the rHA with a concomitant mass increase of 183 Da followed
as a result of reaction with a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl
fluoride (AEBSF).
*Gromadzka B, Smietanka K, Dragun J, Minta Z, Gora-Sochacka A, Szewczyk B. Detection
of changes in avian influenza genome fragments by multitemperature single-strand conformational
polymorphism. Mol Cell Probes. 2008; 22:301-4.
This work was supported by Innovative Economy Operational Program, Grant No. WND-POIG.01.01.02-00-007/08-00
as a part of project “Centre of medicinal product biotechnology. Package of innovative
biopharmaceuticals for human and animal therapy and prophylactics.”
PJ-026
Monoclonal-based antivenomics and biological activities revealing conserved neutralizing
epitopes across elapidae family
Carlos Correa-Netto1,2, Ricardo Araújo1,2, Marcelo Strauch1, Leonora Brazil-Más1,
Marcos Machado3, Moema Leitão-Araújo4, Paulo Melo3, Débora Foguel2, Juan Calvete5,
Russolina Zingali2
1Instituto Vital Brazil, 2Instituto de Bioquimica Medica-UFRJ, 3Programa de Farmacologia
e Química Medicinal-UFRJ, 4Fundação Zoobotânica do Rio Grande do Sul, 5Instituto de
Biomedicina de Valencia-CSIC
Polyclonal antibodies have been used for over a century to the treatment of snakebite
envenoming. New strategies and approaches to understand how antibodies recognize and
neutralize snake toxins represent a challenge to improve the antivenoms. The neurotoxic
activity of Micrurus venom is carried majority by two distinct proteins families,
3FTx and PLA2. The conserved structural folding of these toxins can be appreciated
as model to generate inhibitors against them. In this regard, monoclonal antibodies
(mAbs) can be used as tool to find hot spots for inhibit the toxins and represent
the first step in order to develop recombinant neutralizing molecules. In this work
our goals were analyse a set of monoclonal antibodies against the most toxic components
of M. altirostris venom by proteomics approaches. The venom was fractionated; its
major toxic proteins identified by in vivo tests based on murine lethal toxicity analyses
(approved by the Ethical Committee for Animal Experimentation from Center of Health
and Science of the Federal University of Rio de Janeiro - no. 01200.001568/2013-87).
The toxic components were used to generate a panel of five monoclonal antibodies.
ELISA and antivenomics results allowed us identify the specificity of all mAb and
their neutralizing efficacy was measured by in vitro tests. Three mAbs showed reactivity
towards 3FTx and two against PLA2. All Monoclonal antibodies against 3FTx lack a broad
recognition. However, we identified a pair of monoclonal antibodies able to recognize
all PLA2 molecules of M. altirostris venom and showed a synergism to inhibit the catalytic
activity of them. Moreover, we challenge monoclonal antibodies against to Micrurus
venom for inhibit the PLA2 activity of Naja Naja, specie taxonomically out of Micrurus
cluster. Our results showed that PLA2 of M. altirostris venom share a pair of conserved
antigenic regions and draw attention to use these epitopes to miming antigen to generate
antibodies for antivenom production. Moreover, face to the cross reactivity and the
PLA2 activity inhibition capability by mAbs towards the Naja Naja venom, our results
highlight the conservation of neutralizing epitopes across the Elapidae family.
PJ-027
A comprehensive analysis of scoring functions for protein-protein docking
Didier Barradas1, Juan Fernandez-Recio1,2
1Barcelona Supercomputing Center, 2Joint BSC-CRG-IRB Research Program in Computational
Biology
Protein-protein interactions are known to play key roles in the most important cellular
and biological processes such as signaling, metabolism, and trafficking. One major
goal of structural biology is the structural characterization of all protein complexes
in human and other organisms. These efforts can be complemented by computational approaches.
In this context, computational docking attempts to predict the structure of complexes
from their monomeric constituents. The docking problem presents two main challenges:
the generation of structural poses or sampling, and the identification of the correct
structures with a scoring function (SF). Docking methods can be successful if the
interacting partners undergo small conformational changes. However, in a general situation,
these algorithms generate a large number of incorrect predictions, and therefore the
predictive success strongly depends on the accuracy of the SF used to evaluate the
docked conformations. A variety of strategies have been developed to score putative
protein-protein docked complexes. They are usually based on atomic level potentials,
residue level potentials, or a combination of both. In current work, we have evaluated
73 different SF, taken from Cchappi server, on the results of 3 different rigid body
docking methods, Ftdock, Zdock, and Sdock, using the docking benchmark 4.0 and a docking
set built from CAPRI scorers experiment. Our results show 9 SF that showed better
or similar success rate than the in-built SF. Some of these SF increase the docking
success rates especially for flexible or weak-binding cases, which are the most challenging
for docking. 6 of them are residue level SF robust enough to detected solutions in
cases with large conformation change. In particular we found two SF that shows outstanding
robustness, one designed for protein modeling and shared among docking methods, and
the other is for protein docking which is also the best success rate in the top100
ranking in the CAPRI scorers set. The other 3 atomic level SF display high success
rate to find a solution within weak binding proteins. The 2 most successful SF are
shared between the docking methods and display high success rate in the hard cases
of the benchmark 4.0 and in the CAPRI scorers set. The difference between them in
the resolution level at which they work, one being atomistic the other residue-based.
We found that they success rate vary according to the docking method chosen, allowing
them to explode different properties of the sampling used. This way to characterize
a protein complex can help to develop new combined scoring functions in protein docking
or a new ranking strategy to enhance the success rate.
PJ-028
Multi-PTK antibody: a powerful tool to detect a wide variety of protein tyrosine kinases
(PTKs)
Isamu Kameshita1, Noriyuki Sueyoshi1, Yasunori Sugiyama1
1Kagawa University
The eukaryotic protein kinases consist of large families of homologous proteins and
play pivotal roles in various cellular functions. These enzymes are classified into
two major groups; protein serine/threonine kinases and protein tyrosine kinases (PTKs).
PTKs are believed to be involved in various cellular events such as cell cycle, proliferation,
differentiation, apoptosis, and cell adhesion in multicellular eukaryotes. As many
as 90 PTK genes have been identified in the human genome and many of these PTKs are
known to be closely correlated with various diseases such as cancer. Therefore, it
is important to elucidate the expression profiles of the entire PTK family in cells
and tissues. To investigate the expression profiles of the cellular PTKs, we produced
an antibody that detects a wide variety of PTKs. For production of the antibody, antigenic
peptides corresponding to amino acid sequences of a highly conserved region (subdomain
VIB) of PTKs were synthesized and immunized to BALB/c mice. Among various antigens,
a peptide with 11 amino acids, CYVHRDLRAAN, efficiently produced a polyclonal antibody
with a broad reactivity to PTKs. We established a hybridoma cell line producing a
monoclonal antibody, YK34, which appeared to cross-react with various PTKs. At least
68 PTKs could be detected by YK34 antibody, as evidenced by its reactivity with the
recombinant Src tyrosine kinases whose subdomain VIB had been replaced by those of
the other PTKs. When differentiated HL-60 cells were analyzed by Western blotting
after two-dimensional electrophoresis with YK34 antibody, we observed significant
changes in the immunoreactive spots in HL-60 cell extracts along with the changes
in the morphology of the cells. These results suggest that the Multi-PTK antibody,
YK34, will be a powerful tool for the analysis of a variety of cellular PTKs.
PJ-030
Analysis of the Siglec-9 and hVAP-1 interactions
Leonor Carvalho1, Vimal Parkash1, Heli Elovaara2, Sirpa Jalkanen2, Xiang-Guo Li4,
Tiina Salminen1
1Structural Bionformatics Laboratory, Department of Biosciences, 2MediCity Research
Laboratory, 3Department of Pharmacology, Drug Development and Therapeutics, 4Turku
PET Center
Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglec) are type I transmembrane
proteins. Siglec-9 has an N-terminal V-set domain followed by two C2-set domains in
the extracellular region. It contains an immunoreceptor tyrosinebased inhibitory motif
(ITIMs) in its cytoplasmic tail and can function as an inhibitory receptor by dampening
the tyrosine kinase-driven signaling pathways. These proteins are expressed primarily
on leukocyte subsets and, thus, are thought to be involved in regulation of leukocyte
functions during inflammatory and immune responses. Recently, phage display screening
experiments identified Siglec-9 as leukocyte surface ligand for human vascular adhesion
protein −1 (hVAP-1; AOC3 gene product) and their interaction was confirmed by cell
adhesion and enzymatic assays (Kivi et al., 2009; Aalto et al., 2011). Based on our
preliminary data, hVAP-1 sugar units with sialic acid (SA) might mediate interactions
with the V-set domain in Siglec-9. Furthermore, it is known that the Siglec peptides
binding to hVAP-1 are located in the CE loop of the second C22- set of domain (Siglec-9_C22).
Based on current hypothesis an arginine in Siglec-9_C22 interacts with the TPQ residue
in the active site of hVAP-1. The CE loop of Siglec-9_C22 has two arginines (R284
and R290) and, therefore, the interacting arginine is unclear. We will now study the
interaction mode of hVAP-1 and Siglec-9 in silico to predict the role of the arginines
in the C22 domain and the role of SA-binding using the 3D model of the full-length
ectodomain of Siglec-9 and the hVAP-1 crystal structure. The in silico analysis will
be conducted in parallel with experimental site-specific mutational studies and the
result will be combined to elucidate the mechanism of hVAP-1- Siglec-9 interaction.
PJ-031
Molecular basis of polyubiquitin chain formation by Ube2K
Adam Middleton1, Catherine Day1
1Department of Biochemistry, University of Otago
Attachment of ubiquitin to substrate proteins regulates almost all cellular processes,
including protein degradation and cell division. Ubiquitylation involves a cascade
of three families of proteins: ubiquitin activating (E1), ubiquitin conjugating (E2)
and ubiquitin ligase (E3) enzymes. The 8.5 kDa protein can be attached as a monomeric
moiety or as a polyubiquitin chain, and the type of modification spells out the ’ubiquitin
code’ that directs the fate of the substrate. Polyubiquitin chains can be formed via
eight different linkage types, and the arrangement of chain formation is typically
directed by the E2 enzymes. Forming a polyubiquitin chain involves binding of two
molecules to the E2: the donor (UbD) and acceptor (UbA) ubiquitin. UbD is linked to
the E2 via a thioester bond between its C-terminal Gly and the active site Cys of
the E2, and when primed for catalysis it interacts with a particular face of the E2.
In contrast, coordination of UbA by E2s is transient and cannot be easily measured;
however, UbA binding defines the linkage type of polyubiquitin chains. The E2, Ube2K,
directs Lys48 chain synthesis, which results in modified proteins being degraded by
the proteasome. We generated a stable form of the Ube2K∼Ub conjugate and crystallized
it, and showed that both Ube2K and its ubiquitin conjugate are monomeric. Using molecular
docking, we modelled the position of both UbD and UbA and investigated the interfaces
with site-directed mutagenesis. These experiments led to a molecular model that revealed
how Ube2K can synthesise Lys48-linked ubiquitin chains. This molecular explanation
provides a foundation for understanding how other E2s generate Lys48-linked polyubiquitin
chains.
PJ-032
The two chromophorylated linkers of R-Phycoerythrin in Gracilaria chilensis
Marta Bunster, Francisco Lobos-González, José Aleikar Vásquez, Carola Bruna, José
Martínez-Oyanedl
1Fac de Cs Biol., Universidad de Concepción
The two chromophorylated linkers of R-Phycoerythrin in Gracilaria chilensis. Francisco
Lobos-González, José Aleikar Vásquez, Carola Bruna, José Martínez-Oyanedel, Marta
Bunster. Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas,
Universidad de Concepción. Phycoerythrin is a phycobiliprotein present in phycobilisomes
in Gracilaria chilensis as a complex with chromophorylated linker proteins. Our interest
is to discover the role of these linkers in the function of phycobilisomes. Phycobilisomes(PBP)
are auxiliary light harvesting protein complexes in charge of channeling energy towards
photosystem II in alga, cyanobacteria and cryptophyta. This is possible thanks to
fluorescent proteins called phycobiliproteins (PBP) and the chromophores (phycobilins,
open-chain tetrapyrrols) attached to specific cysteines. Phycobiliproteins share a
common general structure; they are organized as (alfaβ) heterodimers which themselves
assemble as trimers(alfaβ)3 or hexamers (alfaβ)6; this complexes are organized in
high order structures to form the core and the rods. Besides PBPs, PBS have linker
proteins in charge of the assembly and stabilization of the complex, and also it has
been proposed that they collaborate in the fine tuning of the energy transfer steps
between chromophores. These linkers are located within the rods, the rod-core interface,
the core and the core-membrane interface. Although most linker proteins are colorless,
chromophore bearing linkers have been described, which suggest its participation in
the energy transfer process. Two of them, γ 31 and γ 33 are associated to R-phycoerythrin
in Gracilaria chilensis, nevertheless the information available on these linkers in
eukaryots is still limited. To understand how these linkers collaborate with the function
of the phycobilisome, we need structural information, especially the coordinates of
all the chromophores present in the complex; we have sequenced both linkers from the
genomic dna, performed sequence analysis and also we have purified the linkers by
anion exchange, molecular sieve and HPL Chromatography. The characterization was performed
by denaturant electrophoresis, absorption and emission spectroscopy and by mass spectrometry.
The results show that they have molecular masses as predicted, with a peptide signal
for chloroplasts, an internal sequence repeat; residues 67 - 170 with residues 179
– 273 for γ 31 and residues 107-200 with residues 219-315 for γ 33, and the presence
of conserved cysteine residues putative sites of chromophorylation. The spectroscopy
shows that they have different composition of phycobilins and a very short t1/2. A
preliminary model for both linkers shows that they belong to αα structural class and
that they share a common fold (HEAT like motifs) frequently involved in protein-protein
interactions.
Acknowledgement:
FONDECYT 113.0267
PJ-033
Post-docking analysis by physicochemical properties of protein-protein interactions
generated from rigid-body docking processes
Nobuyuki Uchikoga1, Masahito Ohue2, Yuri Matsuzaki3, Yutaka Akiyama3
1Dept. of Phys., Chuo Univ., 2Grad. Sch. of Inform. Sci. and Eng., Tokyo Tech, 3ACLS,
Tokyo Tech
Rigid-body docking algorithms are useful for predicting tertiary structures of near-native
protein complexes. However, this algorithms generate many protein complex poses including
false positives. Then, near-native poses are searched in a post-docking process. There
are many computational softwares with rigid-body docking algorithms, for example,
ZDOCK. We developed a high-performance protein-protein interaction prediction software,
MEGADOCK, which is basically used on supercomputing environments for a large scale
and network level problems in system biology [Matsuzaki, et al. (2013) Source code
Biol. Med. 8:18]. When some large scale network docking analysis, we can use MEGADOCK
by every pairs of proteins for selecting native protein-protein pairs rather than
for prediction of interaction surfaces of native poses. On the other hand, we developed
a profile method for searching near-native protein complexes from docking poses, which
are composed of residues patterns of protein-protein interaction [Uchikoga & Hirokawa
(2010) BMC Bioinform. 11:236]. This profile method is used for exploring more docking
space by clustering interaction surfaces of protein complex poses and iterative docking
processes, which is called ‘re-docking’ [Uchikoga et al. (2013) PLoS ONE 8:e69365].
In this work, we then tried to use these docking softwares and the profile method
for understanding mechanisms of protein-protein interactions. We focused on some physicochemical
properties, electrostatic and hydrophobicity, of a set of protein complex poses generated
by a rigid-body docking process. From these poses, we obtained sets of possible interacting
amino acid pairs. A set of interaction profiles has some information of docking spaces.
From the view of a network prediction, the docking spaces of a set of protein complex
poses are one of the properties for discriminating native protein-protein pairs from
non-native pairs. In this work, ensemble docking process is performed by MEGADOCK
ver. 4.0 and ZDOCK ver. 3.0.1. Cluster analysis is used with profiles of physicochemical
properties. We used a dataset composed of typical 44 monomer-monomer protein pairs
and will discuss mainly differences between native and non-native protein pairs.
PJ-034
The structural studies of the two thermostable laccases from the white-rot fungi Pycnoporus
sanguineus
Marta Orlikowska1, Grzegorz Bujacz1
1Institute of Technical Biochemistry, Lodz University of Technology, Poland
Laccases (EC 1.10.3.2, benzenodiol oxygen oxidoreductases) are enzymes that have the
ability to catalyze the oxidation a wide spectrum of phenolic compounds with the four-electron
reduction of molecular oxygen to water [1]. It has been found that the active site
is well conserved in between laccases from different organisms. It contains four copper
atoms: one paramagnetic type 1 cooper (T1) that is responsible for their characteristic
blue color and where the oxidation of the reducing substrate occurs, one type 2 cooper
(T2) and two type 3 coopers (T3) that conform a trinuclear cluster in which molecular
oxygen is reduced to two molecules of water [2]. Laccases are present in many different
species and they have been isolated from plants, fungi, prokaryotes, and arthropods
In most cases laccases are monomeric glycoproteins of around 500 amino acids with
molecular weights in the range of 60-85 kDa. The various functions carried out by
those enzymes include the antagonistic ones such as their involvement in lignin biosynthesis
(in plants), lignin degradation, pigment production, fruiting body formation, pathogenesis
(in fungi) and spore protection against UV light (in bacteria) [1, 3]. The diversified
functions of laccases make them an interesting enzyme for study from the point of
view of their structure, function and application. Laccases of white-rot fungi (WRF)
are of special interest because one of its role is to degrade lignin and most of them
are extracellular enzymes helping purification procedures [1]. During the last two
decades, there has been an increasing interest in the genus Pycnoporus for its ability
to overproduce high redox potential laccases as the ligninolytic enzymes. We present
the crystal structures of two thermostable lacasses produced by strain Pycnoporus
sanguineus CS43 (LacI and LacII). The molecular weights of LacI and LacII, determined
by SDS-electrophoresis, is 68 and 66 kDa, respectively [3]. Both isoforms shows high
amino acids sequence similarity (91%) between them and high thermal stability, at
50°C and 60°C. They remained active at high concentration of organic solvent (acetonitrile,
ethanol or acetone). The unique properties make them promising candidates for industrial
applications in wasterwater treatment. LacI exerted a higher thermal and pH stability,
tolerance against inhibitors and was a more efficient catalyst for ABTS and DMP (laccases
substrate) then LacII [3]. Based on the structures we would like to understand the
isoforms differences that confers LacI a markedly better performance than LacII in
pH and thermal stability as well as better resistance to inhibitors.
[1] Baldrian F., FEMS Microbiol. Rev., 30, 215–242 (2006)
[2] Mot A. & Silaghi-Dumitrescu R., Biochemistry, 77, 1395-497 (2012)
[3] Rivera-Hoyos E. et al., Fungal Biol. Rev., 27, 67-82 (2013)
[4] Ramirez-Cavazos L. et al., J. Mol. Catal. B Enzym., 108, 32-42 (2014)
PJ-035
Analysis of liver proteome in cystathionine ß-synthase deficient mice using 2D IEF/SDS-PAGE
gel electrophoresis, MALDI–TOF mass spectrometry, and label-free based relative quantitative
proteomics
Izabela Bielińska1, Łukasz Marczak1, Hieronim Jakubowski1,2
1Institute of Bioorganic Chemistry, Polish Academy of Sciences, 2Rutgers University,
New Jersey Medical School
Homocysteine (Hcy) arises from the metabolism of the essential dietary protein amino
acid methionine. Levels of Hcy are regulated by remethylation to Met and transsulfuration
to Cys. Cystathionine β-synthase (CBS) catalyzes the conversion of homocysteine to
cystathionine (first step of transsulfuration reaction). Human CBS deficiency is a
recessive inborn error of homocysteine metabolism that casues severe hyperhomocysteinemia
(HHcy) and diverse clinical manifestations, including fatty liver disease [1]. Although
the causes of fatty liver disease in CBS deficiency have been studied the underlying
mechanism is not understood. We hypothesize that CBS deficiency induces changes in
gene expression that could impair liver homeostasis. To test this hypothesis and gain
insight into hepatic functions of Cbs we analyzed the liver proteome of Cbs -/- and
Cbs +/+ mice [2,3] Using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry
(n=14) we identified twelve liver proteins whose expression was significantly altered
as a result of the Cbs gene inactivation. Expression of three proteins was up-regulated
and of nine down-regulated by the Cbs-/- genotype. Two up-regulated liver proteins
are involved in iron metabolism (Ftl and Fth). Those proteins are associated with
oxidation stress and inflammation. Third up-regulated liver protein (Cbr3) is related
to oxidation-reduction process. The down-regulated protein are involved in the hydrolysis
of N-acylated or N-acetylated amino acids (Acy1), regulation of endopeptidase activity
(A1at4), cholesterol biosynthetic process (Fpps), amino acid degradation (Huth), cellular
calcium ion homeostasis and L-ascorbic acid biosynthetic process (Rgn). Using label-free
based relative quantitative proteomics (n=8) we identified fourteen liver proteins
whose expression was significantly altered as a result of the Cbs gene inactivation.
Expression of four proteins was up-regulated and of ten proteins was down-regulated.
The down-regulated liver proteins are linked with regulation of bone mineralization
and inflammatory response (Ahsg) or regulation of mRNA splicing (Roa2). The up-regulated
liver proteins are involved in tricarboxylic acid cycle (Suca), oxidation-reduction
process (Cy250), cholesterol metabolic process, iron ion homeostasis (Fech), fatty
acid metabolic process (Ssdh; Eci1) and response to oxidative stress (Lonm). Our findings
suggests that Cbs interacts with diverse cellular processes, including lipid metabolism,
that are essential for normal liver homeostasis. Deregulation of genes involved in
lipid metabolism provides a possible explanation for fatty liver disease associated
with CBS deficiency.
[1] Mudd SH, Levy HL, Kraus JP. Disorders of transsulfuration. In Scriver C. R., Beaudet
A. L., Sly W. S., Valle D., Childs B., Kinzler K. W., Vogelstein B., editors. McGraw-Hill;
New York: The Metabolic and Molecular Bases of Inherited Disease. 2001;2:2007–2056
[2] Gupta S et al Mouse models of cystathionine β-synthase deficiency reveal significant
threshold effects of hyperhomocysteinemia. FASEB J 2009;23:883–893
[3] Jakubowski H et al. Genetic or nutritional disorders in homocysteine or folate
metabolism increase protein N-homocysteinylation in mice. FASEB J 2009;23:1721–1727
Supported by the NCN grant 2013/09/B/NZ5/02794, 2013/11/B/NZ1/00091
PJ-037
Investigating protein-protein interactions of the language-related transcription factor
FOXP2 in live cells with bioluminescence resonance energy transfer
Sara B. Estruch1, Sarah A. Graham1, Pelagia Deriziotis1, Swathi Mookonda Chinnappa1,
Simon E. Fisher1,2
1Max Planck Institute for Psycholinguistics, Language and Genetics Department, 2Donders
Institute for Brain, Cognition and Behaviour, Radboud University
Transcription factors play central roles in coordinating developmental processes,
as evidenced by the increasing number of transcription factor-related developmental
disorders being uncovered by next-generation sequencing and genome-wide studies of
copy number variation. The action of a transcription factor in regulating gene expression
depends on interactions with other transcription factors, co-activators/co-repressors
and chromatin modifying and remodeling complexes. Transcription factors are commonly
regulated by post-translational modifications. However the study of protein-protein
interactions and post-translational modifications of transcription factors by common
techniques such as co-immunoprecipitation and mass spectrometry is hampered by the
difficulty in preserving interactions and modifications through cell lysis. To circumvent
this issue, we developed a Bioluminescence Resonance Energy Transfer (BRET) assay,
which allows protein-protein interactions to be observed in live cells. In this assay,
a protein of interest is expressed as a fusion with luciferase from Renilla reniformis,
and its putative interaction partner as a fusion with Yellow Fluorescent Protein (YFP).
Upon addition of a cell-permeable substrate, the distance-dependent non-radiative
transfer of energy from luciferase to YFP is quantified by measurement of light emission
at two wavelengths to assess the interaction between the two fusion proteins. To validate
the utility of this assay for investigating transcription factor interactions, we
confirmed homodimerization of the FOXP2 transcription factor, haploinsufficiency of
which causes a rare and severe speech and language disorder, as well as interaction
of FOXP2 with other members of the FOXP family. We also confirmed the interaction
between FOXP2 and multiple candidate interactors identified through yeast two-hybrid
assays, including the autism-related transcription factor TBR1, the co-repressors
CtBP1 and CtBP2, and post-translational modification enzymes of the PIAS family. The
role of PIAS enzymes in sumoylation – the covalent modification of proteins with Small
Ubiquitin-like Modifier (SUMO) proteins – led us to further explore this process,
which is notably difficult to investigate because of the dynamic and labile nature
of the modification, which is also typically present on only a minor fraction of molecules
of a given protein. Combining the BRET assay with gel-shift techniques we demonstrated
that FOXP2 is sumoylated. Finally, we used the BRET assay to examine the effects of
etiological FOXP2 variants in speech and language disorder on protein-protein interactions
and post-translational modification. In summary, the BRET assay is a sensitive, reliable
and potentially high-throughput technique for exploring protein biology in the context
of live cells. We have demonstrated applications of the assay in validating putative
protein-protein interactions, assessing post-translational modifications, and investigating
functional effects of protein variants identified in patient cohorts. These investigations
have provided novel insights into the function of the FOXP2 transcription factor in
neurodevelopment and into the etiology of FOXP2-related speech and language disorder.
PJ-038
The directly interaction between PreS1 of human virus B and human heat shock protein
70 (HSP70)
Deqiang Wang1, Chen Ke1, Jun Zhang2
1Key Laboratory of Molecular Biology on Infectious Disease, 2The Department of Cell
Biology and Genetics
The directly interaction between PreS1 of Human virus B and Human Heat Shock protein
70 (HSP70). Hepatitis B virus (HBV) has infected 2 billion people worldwide, and 350
million of them are chronically infected. The chronic virus infection, a major public
health problem worldwide, leads to bout two-thirds of hepatocellular carcinoma (HCC).
The HBV envelope consists of the large (L), middle (M) and small (S) envelope proteins,
which contain preS1-preS2-S, preS2-S, and S domain alone, respectively [2]. The preS1
domain is believed to mediate virus attachment to the high-affinity receptor. Yan
et al employed a novel technique to propose sodium taurocholate co-transporting polypeptide
(NTCP) as the candidate HBV receptor, and consequently, NTCP is a target for a new
family of anti-HBV agents [3]. Whereas, it remains a query to clarify that NTCP is
the only or major HBV receptor in vivo. To illuminate if other host proteins cooperatively
participate the HBV infection, we detect the interaction between PreS1 and many candidate
host proteins. Fortunately, we have found that the human heat shocking protein 70
(HSP70) could directly interact with the PreS1 domain of the HBV virus protein. Both
the pull down and the size exclusion chromatography experiments verify that the GRP78
have the ability binding to PreS1. Whereas, whether the interaction between HSP70
and PreS1 relates to the HBV infection need further experiments to clarify.
Reference:
[1] Lavanchy. J. Viral Hepat. 2004, 11: 97–107.
[2] Schulze et al. Hepatology 2007, 46: 1759–1768.
[3] Yan et al., Elife 2012, 1: e00049
PJ-039
A new hydrophobicity scale of amino acids based on IEF-MST calculated log P and log
D
William J. Zamora1, Josep M. Campanera1, F. Javier Luque1, Jody McGinness1
1Departament de Fisicoquímica and Institut de Biomedicina (IBUB), 2Departament de
Fisicoquímica and Institut de Biomedicina (IBUB), 3Departament de Fisicoquímica and
Institut de Biomedicina (IBUB), 4The member sponsorship
In the vast world of naturally occurring peptides, where more than 7000 peptides are
known and approximately 140 peptide therapeutics are currently being evaluated in
clinical trials (Fosgerau & Hoffmann, 2015), the rapid and accurate determination
of their physicochemical properties is key in peptide drug discovery. Among these
properties, hydrophobicity is crucial for understanding molecular recognition and
biomolecular aggregation. Hence, there is a great interest in determining hydrophobicity
scales for amino acid structures. In this work, octanol/water partition (log P) and
octanol/water distribution (log DpH, Fig. 1) of N-acetyl-L-amino-acid methyl amides
were determined by means of quantum mechanical IEF-MST solvation calculations taking
into account the intrinsic conformational preferences of each amino acid according
to Dunbrack's libraries (Dunbrack & Karplus, 1993;1994). The results reveal log D7.4
differences for α-helical and β-sheet conformations in Arg, Lys, Hid, Asn, Gln, Met,
Cys, Leu and Ile. Furthermore, by decomposing the octanol/water transfer free energy
into electrostatic and non-electrostatic components, we estimated that the non- electrostatic
cost of transferring the amino acid side chain amounts to 23.9 ± 3.0 cal/mol.Å2, in
agreement with previous estimates reported in the literature. Comparison of our scale
with other theoretical and experimental hydrophobicity scales yields satisfactory
results, leading to correlation coefficients ranging from 0.61 to 0.94. Additionally,
the MST-derived hydrophobicity scale led to significant correlations with the RP-HPLC
retention factors measured for eight decapeptides (r = 0.97) and for 195 influenza
virus hemagglutinin 13-mer (Ac-YPYDVPDYASLRS-Amide) peptides (r = 0.80). Finally,
the hydrophobicity scale was able to reproduce the experimental log P for 118 random
neutral peptides (r = 0.92) and log D7.4 for ’01:15 random charged peptides (r = 0.95),
Fig. 2. Future studies will address the application of this methodology to nonproteogenic
amino acids, the prediction of peptide hydrophobicity at global and atomic level in
peptides, and the scoring of peptide-protein interactions.
Figure 1.
Representation of log D7.4
values for twenty-one amino acid species. Black circle, red box and blue triangle
represent the log D7.4
value in total, alpha-helix and beta-sheet conformers respectively. Residues in bold
show different values between alpha-helix and beta-sheet conformers
Figure 2.
Representation of experimental log D7.4
for 115 random peptides versus a) computed hydrophobicity using our log P values (correlation
in yellow) b) computed hydrophobicity using our log D7.4
values (correlation in red).
PJ-040
Docking-based tools for discovery of protein-protein modulators
Mireia Rosell Oliveras1, Juan Fernández Recio2
1Barcelona Supercomputing Center, 2Barcelona Supercomputing Center
Docking-based tools for discovery of protein-protein modulators. Protein-protein interactions
(PPIs) play an essential role in many biological processes, including disease conditions.
Strategies to modulate PPIs with small molecules have therefore attracted increasing
interest over the last few years. Although protein-protein interfaces (PPIfs) are
considered difficult to target with small molecules given its lack of well defined
cavities. Successful PPI inhibitors have been reported into transient cavities from
previously flat PPIfs. Recent studies emphasize on hotspots (those residues contribute
for most of the energy of binding) as promising targets for the modulation of PPI.
PyDock algorithm is one of the few computational methods that use energy of solvation
to predict protein-protein interfaces and hotspots residues. We present an approach
aimed at identifying hotspots and transient pockets from predicted protein-protein
interfaces in order to find potential small molecules capable of modulating PPIs.
The method uses pyDock to identify PPIfs and hotspots and molecular dynamics (MD)
techniques to propose putative transient cavities. We benchmarked the protocol in
a small set of protein-protein complexes for which both structural data and PPI inhibitors
are known. The method applies to the unbound proteins of the complexes the fast Fourier
transform algorithm, followed by the energy-based scoring from pyDock to calculate
the normalized interface propensity (NIP) values derived from rigid-body protein docking
simulations to predict the PPIfs and hotspots residues without any prior structural
knowledge of the complex. Then we used MD to describe the possible fluctuations of
the interacting proteins in order to suggest transient pockets that could be useful
as targets of small molecules for the modulation of PPIs. Finally, we evaluated by
ligand docking, the validity of predicted hotspots and pockets for in silico drug
design. We found that the NIP-based method from pyDock protein-protein docking identifies
hotspots residues that are located within the binding site of known inhibitors of
PPIs. Predicting PPIfs from a three dimensional structure is a key task for the modulation
of PPIs. The use of the NIP-based hotspots prediction method improve the identification
of transient cavities from MD simulation when compared to known binding cavities.
This approach can be extremely useful in a realistic scenario of drug discovery targeting
PPIfs, when there is no information at all about the protein-protein complex structure.
PJ-041
Identification of transient protein complexes by using intrinsic disorder and network
topology
Inhae Kim1, Sangjin Han1, Jihye Hwang2, Sanguk Kim1
1Department of Life Sciences, Pohang University of Science and Technology, 2Division
of IT Convergence Engineering, Pohang University of Science and Technol
Protein complexes are the fundamental molecular organizations that assemble multiple
proteins to achieve various biological processes. Identification of protein complex
membership should provide a genotype-phenotype map to elucidate human gene-disease
associations. It has been routinely assumed that network clusters with dense connections
inside and sparse connections outside would form functional protein complexes. Therefore,
searching highly modular subgraphs in protein-protein interaction networks was explicitly
or implicitly implemented in the algorithms to find protein complexes. However, to
our surprise, we found a large portion of complexes with a medium-to-low modularity
from the analysis of 719 experimentally confirmed protein complexes. We also discovered
that these complexes have cellular functions enriched in highly time- and space-dependent
expression, such as signal transduction or subcellular localization. We further developed
an algorithm to find such complexes by weighing network connections to capture transient
interactions with intrinsically disordered regions. We confirmed that our method improved
the identification of biologically relevant members of protein complexes and covered
more complexes with a medium-to-low modularity. Furthermore, newly discovered subunits
in protein complexes could explain more disease-gene associations, indicating its
utility to expand current genotype-phenotype map of human diseases.
PJ-042
Expanding template-based protein-protein complex prediction using ab-initio docking
Sergio Mares-Sámano1, Luis Ángel Rodríguez-Lumbreras1, Juan Fernández-Recio1
1Joint BSC-CRG-IRB Research Program in Computational Biology
Structural characterization of protein-protein interaction (PPI) networks is crucial
for understanding the underlying molecular mechanisms whereby life processes and disease
arise. However, due to inherent limitations of experimental techniques, such characterization
only covers an extremely reduced fraction of the human PPI network (interactome).
Recent studies have shown that although available structural templates may suffice
to model a significant proportion of the interactome, model accuracy and binding specificity
remain unsolved problems. Consequently, improving the ability to predict PPIs structurally
will help to provide a better 3D profile of the known interactome, which may ultimately
lead to the development of new therapeutic applications. Here we show a novel approach
that combines template-based modeling with protein-protein computational docking to
the structure-based prediction of PPIs. Our approach samples different protein-protein
structural models derived from docking simulations. Models are subsequently ranked
using a function that incorporates an energy-based scoring term and a structural template
similarity score. The energy-based scoring function includes electrostatics, van de
Waals and desolvation calculations, whilst the template similarity score accounts
for the degree of structural similarity of models against a high-resolution and diverse
dataset of structural templates. Our approach highly improved the predictive success
rate over individual ab-initio docking and template-based techniques across a large
benchmark dataset, including 176 protein-protein complexes. When compared to the performance
of the ab-initio docking algorithm, we found that the approach increased consistently
the success rate, by approximately 30%, for the top 1, top 5 and top 10 solutions.
The success rate improvement was even more notorious when the comparison was performed
against the predictions from the traditional template-based docking. Though incorporating
ab-initio docking expands considerably the scope of the template-based docking method,
challenges remain for interacting proteins in which high conformational changes occur
upon binding and also the size and diversity of the repertoire of structural templates
needs to be increased.
PJ-043
A Common Role for Cytochrome c in Programmed Cell Death in Humans and Plants
Katiuska González-Arzola1, Blas Moreno-Beltrán1, Jonathan Martínez-Fábregas1, Carlos
A. Elena-Real1, Antonio Díaz-Quintana1, Irene Díaz-Moreno1, Miguel Á. De la Rosa1
1 Instituto de Bioquímica
A Common Role for Cytochrome c in Programmed Cell Death in Humans and Plants
González-Arzola, Katiuska; Moreno-Beltrán, Blas; Martínez-Fábregas, Jonathan; Elena-Real,
Carlos A.; Díaz-Quintana, Antonio; Díaz-Moreno, Irene; De la Rosa, Miguel Á.
Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartuja
Universidad de Sevilla – CSIC, Sevilla, Spain
Email: katiuska.gonzalez@ibvf.csic.es
Programmed Cell Death (PCD) is essential for the development of multicellular organisms.
In mammalian cells, early events in PCD involve the release of cytochrome c (Cc) from
mitochondria to the cytoplasm, so letting Cc play a key role in assembling the apoptosome
and triggering apoptosis. In plants, PCD is part of a general process – the so-called
hypersensitive response – in which mitochondrial Cc is likewise released into the
cytosol but its further role and cytoplasmic partners remain veiled. Such a coincidence
in Cc release made us think of a common link for PCD in such evolutionarily distant
species along evolution.
To go deeper in understanding the PCD-dependent role of Cc, a proteomic approach based
on affinity chromatography with Cc as bait was run using human and plant cell extracts.
Upon combining this approach with Bimolecular Fluorescence Complementation (BIFC),
a total of eight and nine unknown proteins interacting with Cc under PCD conditions
were identified in human and plant cells, respectively [1,2]. Such novel Cc-partners
– which are located in the cytoplasm and even in the nucleus – are involved in protein
folding, translational regulation, oxidative stress, DNA damage, energetic and mRNA
metabolism [3]. Strikingly, some of the novel human Cc-partners are closely related
to those for plant Cc, so indicating that the evolutionarily well-conserved event
of Cc release from mitochondria could involve a common signalosome consisting of a
wide range of common targets [3].
To also understand such a promiscuity of Cc from a structural point of view, the Cc
surface residues involved in complex formation with each one of its counterparts were
mapped by using NMR spectroscopy. The resulting data shows that the heme crevice of
Cc is at the Cc-partner interface in most of the complexes, which is in agreement
with the vast majority of known redox adducts of Cc. In contrast, however, to the
high turnover number of the redox Cc adducts inside the mitochondria, the complexes
formed by Cc under PCD conditions lead to the formation of rather stable nucleo-cytoplasmic
ensembles.
Altogether, these findings suggest that extra-mitochondrial Cc interacts with nuclear
and/or cytoplasmic pro-survival, anti-apoptotic proteins in both humans and plants
so as to lead living cells to dye.
Keywords:
Cytochrome c, Programmed Cell Death, Signalosome.
[1] Martínez-Fábregas J., Díaz-Moreno I., González-Arzola K., Janocha S., Navarro
J.A., Hervás M., Bernhardt R., Díaz-Quintana A., De la Rosa M.A. (2013). Mol. Cell.
Proteomics, 12: 3666-3676.
[2] Martínez-Fábregas J., Díaz-Moreno I., González-Arzola K., Janocha S., Navarro
J.A., Hervás M., Bernhardt R., Velázquez-Campoy A., Díaz-Quintana A., De la Rosa M.A.
(2014). Mol. Cell. Proteomics, 13: 1439-1456.
[3] Martínez-Fábregas J., Díaz-Moreno I., González-Arzola K., Díaz-Quintana A., De
la Rosa M.A. (2014). Cell Death Dis., 5: e1314.
PJ-044
Phosphorylation of cytochrome c at positions 28 and 47 could affect its double role
in the cell
Alejandra Guerra-Castellano1, Katiuska González-Arzola1, Francisco Rivero-Rodríguez1,
Adrián Velázquez-Campoy2, Miguel Ángel De la Rosa1, Irene Díaz-Moreno1, Antonio Díaz-Quintana1
1IBVF - cicCartuja, University of Seville - CSIC, 2BIFI-IQFR, University of Saragossa
– CSIC
Post-translational phosphorylation often modulates the function of proteins. In particular,
they affect the role that cytochrome c (Cc) plays in cell life and death [1]. Cc is
phosphorylated in vivo in Tyr48 and Tyr97 residues [2, 3], but recently, two new phosphorylation
sites have been described at positions 28 and 47 [4]. Hence, we aim at understanding
the structural and functional changes induced by Thr28 and Ser47 phosphorylation Cc.
For this purpose, we designed two phosphomimetic mutants of Cc by replacing either
Thr28 or Ser47 by the canonical amino acid aspartic acid (T28D and S47D). As control,
two other mutants at the same two positions (T28A and S47A) were analyzed so as to
differentiate the effects due to the presence of a negatively charged residue. Remarkably,
the S47A mutant is significantly less stable than the wild-type species. We found
that phosphorylation at position Thr28 diminishes the redox potential and oxygen consumption.
In addition, T28D mutation affects the ability of Cc to bind the distal site pCc1,
thereby suggesting that phosphorylation at this position affects the electron carrier
capacity of Cc.
Work supported by JAE Program (JaePre_2011_01248), ESF 2007-2013, Andalusian Government
(CVI-BIO198), Ministry of Economy and Competitiveness (BFU2012-31670).
1 García-Heredia, JM et al. J. Biol. Inorg. Chem. (2011) 16, ’01:155-1168
2 Lee, I et al. Biochemistry (2006) 45, 9121-9128
3 Yu, H et al. Biochim. Biophys. Acta (2008) 1777, 1066-1071
4 Zhao, X et al. Mol. Cell. Proteomics. (2011) 10, 1-14
PJ-046
msBiodata Analysis Tool, a web tool to extract relevant information from proteomics
experiments
Pau Marc Muñoz Torres1, Robert Baluzic1, Ivana Grebesa1, Oliver Vugrek1
1Translational Medicine Group
Mass spectrometry (MS) is widely used techniques to gain knowledge about biomolecules
[1, 2]. It produces a high amount of data which is often presented as a list containing
thousands of proteins. That list usually contains few hits interesting for our research.
The pocess to select those proteins may include integrating experimental with annotation
data. It requires spending some time in both, performing calculus and searching in
databases. In this poster we present msBiodata Analysis Tool, a web service thought
to deal with this tedious work. With this tool, researchers can set rules to select
the most interesting hits in his lists using both, experimental data and Gene Ontology
[3] annotation. The data can be upload to the web using an excel spreadsheet or a
flat files in a mztab format, and rules are easily constructed by means logical sentences.
Those sentences are composed by one or more terms linked by logic operators (and and
or). Each term in the logical sentence indicates to our program the conditions 1 that
selected hits must meet. Once the alysis is finished, the results are delivered by
e-mail. msBiodat analysis tool do not requires any programming knowledge to be used
and is freely available at: http://msbiodata.innomol.eu Keywords Bioinformatics/Data
analysis/proteomics/Data mining/Mass spectrometry.
References:
[1] Kondethimmanahalli Chandramouli and Pei-Yuan Qian. Proteomics: challenges, techniques
and possibilities to overcome biological sample complexity. Hum Genomics Proteomics,
2009, 2009.
[2] Bruno Domon and Ruedi Aebersold. Mass spectrometry and protein analysis. Science,
312(5771):212–217, Apr 2006.
[3] M. Ashburner, C.A. Ball, J.A. Blake, D. Botstein, H. Butler, J. M. Cherry, A.
P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, M. A. Harris, D. P. Hill, L. Issel-Tarver,
A. Kasarskis, S. Lewis, J. C. Matese, J. E. Richardson, M. Ringwald, G. M. Rubin,
and G. Sherlock. Gene ontology: tool for the nification of biology. the gene ontology
consortium. Nat Genet, 25(1):25–29, May 2000.
PK-001
Effect of three aporphine alkaloids on bacillus subtilis 168
Fatma Gizem Avci1, Berna Sariyar Akbulut1
1Marmara University, Department of Bioengineering
Extensive and misuse of antimicrobial drugs have generated selective pressure on bacteria
which resulted in the development of various resistance mechanisms rendering the drugs
ineffective. Increased resistance is a matter of serious public health concern worldwide.
Discovery and development of new antimicrobials are crucial steps to overcome bacterial
resistance. In this sense, plant-derived substances have drawn attention as novel
and promising sources of antimicrobial agents. Alkaloids are among the secondary metabolites
of plants with different activities. So far, isoquinoline alkaloids have proven to
possess therapeutic potential. In this work, the effect of three aporphine alkaloids,
boldine, bulbocapnine and roemerine, on the microbial viability has been investigated
using a systems approach. The cells were allowed to grow in the presence of the alkaloids
for determining MIC values. Cytoscape was then used to analyze protein interaction
networks. Despite their structural similarities, whereas -(-)roemerine had a high
antimicrobial activity against Bacillus subtilis 168, boldine and bulbocapnine had
no significant effect. The network constructed using cytoscape enabled us to evaluate
the protein interactions comparatively under the influence of the three different
alkaloids. -(-)Roemerine is a potential antibacterial agent that could be valuable
in the fight with rising resistance to available drugs.
Acknowledgements:
This work was supported by TUBITAK-MAG project 113M052.
PK-002
Protein degradation systems in the control of salmonid fish growth
Liudmila Lysenko1, Nadezda Kantserova1, Marina Krupnova1, Nina Nemova1
1The Institute of Biology, Karelian Research Centre of Russian Academy of Science
Beside the rate of protein synthesis, the regulation of protein degradation plays
a crucial role in the white muscle protein accumulation and overall fish growth. Intracellular
proteolysis in salmonid species, such as Atlantic salmon, Salmo salar L. and rainbow
trout, Oncorhynchus mykiss Walb., was studied to evaluate the basic mechanisms of
protein degradation that could possess a potential target to regulate the body mass
accumulation in farmed fish. A number of white muscle proteases such as cathepsins
B, L, and D, proteasomes, and calcium-dependent proteases (µ- and m-calpains), was
studied in the juvenile specimens of different size- and age-groups both wild and
farmed salmonids. The correlations between the protease activity and expression levels
and morphometric characteristics of fish were found. The size- and age-related differences
in intracellular protease activity revealed in fish muscles indicate both general
role of proteolysis regulation in salmonid growth and the specific role of the individual
proteolytic enzymes as well. The data on negative correlation of cathepsin D and calpain
activity in muscles and the rate of weight increase in juvenile salmonids were obtained.
A revealed positive correlation of cathepsin B activity and morphometric parameters
in fish young presumably indicates its primary contribution to non-myofibrillar protein
turnover. Ubiquitin-proteasome system seems to contribute to background protein turnover
as the proteasome activity was not corresponded with growth rate. Summarizing the
data obtained the autophagy-lysosomal and calpain-related protein degradation pathways
were recognized to be directly involved in body growth and muscle protein retention
in salmonid fish. The work was carried out using technical facilities of IB KarRC
RAS Equipment Centre and financially supported by the Russian Science Foundation,
grant No. 14-24-00102 “Salmonids of the North-West Russia: ecological and biochemical
mechanisms of early development”.
PK-003
Solving the proteomic organization of fitness-related genes in Uropathogenic Escherichia
coli
Marc Torrent Burgas1,2
1Microbiology Department, Vall d’Hebron Institut de Recerca, 2Biochemistry Department,
Universitat Autònoma de Barcelona
Certain isolates of Escherichia coli are associated to a wide range of diseases and
affect both humans and animals worldwide. Uropathogenic E. coli (UPEC) belongs to
a subset of the extraintestinal pathogenic E. coli (ExPEC) pathovar and is the primary
etiological agent of urinary tract infections (UTI). Patients with UTI can develop
pyelonephritis and are at risk for developing bacteremia that may result in life threatening
sepsis. Nowadays, complete genomes for almost all major bacterial pathogens are available,
helping researchers to identify virulence factors. However we still ignore how these
genes are organized at the proteome level and how this association influences bacteria
pathogenicity. We integrated available databases on UPEC E. coli (strain CFT073) to
investigate the genomic and proteomic organization of genes related to UPEC fitness
in the host. Intriguingly, we found that most fitness-related genes have orthologs
not only in other pathogenic strains but also in non-pathogenic bacteria such as E.
coli K-12. These genes are organized in clusters and operons with similar structure.
By integrating protein-protein interaction data we observed that genes with high impact
on fitness also display a highly clustered organization when compared to other genes.
Overall, our results show that protein-protein interaction clusters associated to
UPEC fitness in the host represent a promising target for the design of new antibiotics.
PK-004
Elucidating the molecular mechanisms by which the HNH endonuclease gp74 activates
the terminases in bacteriophage HK97
Sasha Weiditch1, Karen Maxwell4, Voula Kanelis1,3
1Cell & Systems Biology, University of Toronto, 2Donnelly Centre for Cellular and
Biomolecular Research, University of Toronto, 3Chemical & Physical Sciences, University
of Toronto, 4Department of Molecular Genetics, University of Toronto
Elucidating the molecular mechanisms by which the HNH endonuclease gp74 activates
the terminases in bacteriophage HK97. Bacteriophages are the most abundant entities
in the biosphere (1). The last gene in the genome of the bacteriophage HK97 codes
for gp74, an HNH endonuclease(2). HNH endonucleases are characterized by two highly
conserved His residues and an Asn residue(3). Gp74 is essential for phage head morphogenesis,
likely because gp74 enhances the activity of the HK97 terminase enzymes toward the
cos site(4). Notably, enhancement of the terminase-mediated cleavage of the phage
cos site requires the presence of an intact HNH motif in gp74. Mutation of the canonical
metal binding His in the HNH motif abrogates gp74 mediated-terminase activity. Although
phages are widely studied, there is no definitive structural or mechanistic evidence
as to how the HNH endonuclease within gp74 functionally interacts with the adjacent
terminase enzymes to facilitate phage morphogenesis. Previous work on HNH-containing
bacteriophage proteins does not address explicitly how the requirement for divalent
metal binding at the HNH endonuclease site induces interaction with the terminase
enzymes that are so crucial for phage DNA packaging during morphogenesis (4, 5). In
addition, gp74 possesses no sequence similarity to HNH proteins for which the structure
has been determined (3), making structural studies of gp74 necessary. Toward these
ends, we use nuclear magnetic resonance (NMR) spectroscopy to probe metal and terminase
binding of gp74 in the wild type state and bearing metal binding mutations. We also
report backbone resonance assignment of gp74. Our NMR studies have elucidated residues
within gp74 required for metal binding and terminase activity. These data are being
used to assess the role of specific gp74 residues in phage morphogenesis. Together,
this work will identify the enigmatic role describing how metal binding in HNH endonucleases
is crucial in the replication and morphogenesis of phages.
1. Campbell, A. (1994) Comparative molecular biology of lambdoid phages, Annu Rev
Microbiol 48, 193-222.
2. Juhala, R. J., Ford, M. E., Duda, R. L., Youlton, A., Hatfull, G. F., and Hendrix,
R. W. (2000) Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic
mosaicism in the lambdoid bacteriophages, J Mol Biol 299, 27-51.
3.Stoddard, B. L. (2005) Homing endonuclease structure and function, Q Rev Biophys
38, 49-95.
4. Kala, S., Cumby, N., Sadowski, P. D., Hyder, B. Z., Kanelis, V., Davidson, A. R.,
and Maxwell, K. L. (2014) HNH proteins are a widespread component of phage DNA packaging
machines, Proc Natl Acad Sci U S A 111, 6022-6027.
5.Quiles-Puchalt, N., Carpena, N., Alonso, J. C., Novick, R. P., Marina, A., and Penades,
J. R. (2014) Staphylococcal pathogenicity island DNA packaging system involving cos-site
packaging and phage-encoded HNH endonucleases, Proc Natl Acad Sci USA 111, 6016-6021.
PK-005
Analysis of the binding of mycotoxins to proteins involved in ASD with a combined
computational/experimental approach
Bernardina Scafuri1, Antonio Varriale2, Angelo Facchiano2, Sabato D’Auria2, Maria
Elisabetta Raggi3, Anna Marabotti1
1Dept. Chemistry and Biology, University of Salerno Bernardina Scafuri, 2Institute
of Food Science, CNR Antonio Varriale, 3IRCCS “E. Medea” Ass. “La Nostra Famiglia”
Maria Elena Raggi, 4.−2 Sabato D’auria, 5.−2 Angelo Facchiano, 6.−2 Anna Marabotti,
7.−1 Anna Marabotti
Autism spectrum disorder (ASD) is a group of neurodevelopmental disabilities characterized
by persistent deficits that manifestwith impaired social communication and social
interaction, restricted and repetitive patterns of behavior, interests or activities
[1]. The etiology of ASDis unknown, but it is believed that it involves genetic and
environmental components. The purpose of this work is to assess the possible involvement
of food contaminants, such as mycotoxins, in the etiology of ASD. The hypothesis is
that the mycotoxins ingested with the diet could bind to proteins and expose the entire
organism,including CNS, to the negative effects of xenobiotics, in genetically predisposed
patients. In this study some possible protein targets for the mycotoxinswere identified
to evaluate if the bond between any protein target and the mycotoxin in exam could
play a role in ASD. Twelve mycotoxins were selected (ochratoxin A, gliotoxin, aflatoxin
B1, aflatoxin B2, aflatoxin M1, aflatoxin M2, aflatoxicol, a-zearalanol, b-zeralanol,
zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals.For
each of these molecules,possible protein targets were searched by a reverse docking
approach using the idTargetserver[2].From the results given by idTarget, human protein
targets expressed in the brain or involved in brain diseaseswere selected. Subsequently,
a direct docking was made using AutoDock 4.2 [3], in orderto verify the strength of
the interaction between selected proteins and each mycotoxin, and to identify the
mycotoxins’ binding site on each of the selected protein. Finally, the bond of some
mycotoxins to selected protein targets has been experimentally tested. For each mycotoxin,
idTarget returned thousands of possible protein targets,and only those with the best
binding energy were selected and evaluated. Among them, human protein targets that
are expressed in the brain or that are involved in cerebral diseases,have been selected;
moreover the protein targets that were not human but that idTargetselected for five
or more mycotoxins, were replaced with their human counterparts. At the end of the
procedure, nineteen protein targets have been identified for the following direct
docking approach. From the docking results, eight proteins have been selected for
experimental tests, having a predicted binding energy lower than −7 kcal/mol. Finally,
the interactions between Acetylcholinesterase (AChE), β-secretase (BACE1) and Neuroligin-4,
X-linked (NLG4X) with Aflatoxin B1, Aflatoxin B2, Gliotoxin, Ochratoxin A and Deoxynivalenol,
were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. These
experiments confirmed the presence of an interaction between BACE1 and Aflatoxin B1;
NLG4X and Aflatoxin B1,Gliotoxin and Ochratoxin A; and Deoxynivalenol,AChE and Aflatoxin
B1. These results suggest that the interaction between mycotoxins and proteins involved
in neuronal plasticity is possible also in vivo, supporting the hypothesis of a putative
role of mycotoxins in the etiology of ASD.
References:
[1] Singh K, Zimmermann AW;Sleep in Autism Spectrum Disorder and Attention Deficit
Hyperactivity Disorder; SeminPediatrNeurol 22,113-125 (2015)
[2]Chu PY, Chen CM, Lin JH;idTarget: a web server for identifying protein targets
of small chemical molecules with robust scoring functions and a divide-and-conquer
docking approach, Nucleic Acids Res 34, W219-W224 (2006)
[3]Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ;AutoDock4
and AutoDockTools4: Automated docking with selective receptor flexibility, J ComputChem
30, 2785-2791 (2009)
Acknowledgements:
This work was made in the frameshift of the project GR-2009-1570296: “The relationshipamongfood,
mycotoxins, gastrointestinaldisorders and autism: a multidisciplinaryapproach for
the molecularinvestigation” funded by the Ministry of Health (program “Ricerca Finalizzata
e Giovani Ricercatori 2009”). AF acknowledges the contribution of FlagshipInterOmics
Project (PB.P05, funded and supported by the ItalianMinistry of Education, University
and Research and Italian National ResearchCouncilorganizations)
The Authors would also acknowledge the support of NanoTemper Technologies GmbH, and
in particular of dr. Francois-Xavier Ogi, for the microscale thermophoresis experiments
PK-006
Developing of microbial consortia for enzymatic valuable conversion of keratin-rich
slaughter-house waste
Roall Espersen1, Milena Gonzalo3, Samuel Jacquiod3, Waleed Abu-Alsud3, Søren J. Sørensen3,
Jakob R. Winther4, Per Hägglund2, Birte Svensson1
1Enzyme and Protein Chemistry, Department of Systems Biology, Technical Universit,
2Protein and Immune Systems Biology, Department of Systems Biology, Technical Uni,
3Section of Microbiology, Department of Biology, University of Copenhagen, 4Section
for Biomolecular Sciences, Department of Biology, University of Copenhag
Meat production from pigs for human consumption is a resource heavy process, indeed
every part of the animal that is not used constitutes a protein food-chain loss, which
is neither economically nor environmentally viable. The goal of this project is to
better harness slaughterhouse waste such as the keratin rich pig bristles and nails
through microbial conversion. Instead of using identified single microorganisms, it
is the goal to define microbial consortia where microorganisms synergistically show
the ability of efficient keratin degradation/conversion. Candidate consortia have
been obtained by selecting for microorganisms growing on enriched media that contains
milled pig bristles as sole carbon and nitrogen source. By using mass spectrometry
and various biochemical analyses to investigate keratinolytic enzymes, methods will
be established for identifying and characterizing suitable consortia. Protein families
likely to be involved are keratinases, which are specialized proteases including serine,
cysteine and metallo proteases, as well as systems capable of reducing or otherwise
breaking disulfide bonds which are highly abundant in hair and nails. Furthermore,
interactions and symbiosis of microorganisms in a consortium will be investigated
at the meta-proteomics level. The project will lead to development of biotechnological
degradation of keratin rich fibers, and provide new insights into functional dynamics
and efficacy of microbial consortia.
PK-007
A comprehensive protein domain analysis to map cancer-type-specific somatic mutations
Jihye Hwang1, Sangjin Han2, Inhae Kim2, Sanguk Kim2
1Department of IT Convergence and Engineering, POSTECH, 2Department of Life Science,
POSTECH
Recent development of whole genome sequencing technology has provided a large amount
of genetic variants of cancers. This data may be informative to find cancer markers
for diagnosis and stratification of cancer patients into different risk groups. However,
the identification of type-specific markers for various cancers is still elusive because
each genetic variant often accounts for only small portion of patients. Thus, it is
urgent to develop analytic frameworks to infer the genotype-phenotype relationship
in cancers from such amount of genetic variants. In this study, we mapped protein
domain information to genetic variants from whole-genome/exome sequencing data of
3,527 specimens across 12 cancer types from The Cancer Genome Atlas project (TCGA
project). We found that protein domain instances are often enriched with cancer-type-specific
somatic mutations, which enabled us to define cancer-type-specific protein domains.
Based on the cancer-type-specific protein domains, we could stratify cancers with
distinct phenotypes better than gene-based approaches. Thus, we expect that protein
domain analysis offers a new opportunity to decipher functional relationships between
cancers and would helpful to prioritize cancer-type-specific mutations for discovering
marker genes and classification of cancer patient groups.
PK-008
Identification of cancer-type-specific modules comprised of cancer-type-specific variants
through phenotype similarity between cancer types
Sangjin Han1, Jihye Hwang2, Inhae Kim1, Sanguk Kim1,2
1Department of Life Science, POSTECH, 2Department of IT Convergence and Engineering,
POSTECH
Interpretation of the genome-wide association studies (GWAS) of cancer patients to
find cancer-type-specific biomarker is challenging due to the mutational heterogeneity
of cancer types. Network approaches to find cancer-type-specific variants and biological
pathways are increasing since genes tend to act together to display phenotypic or
disease outcomes. Phenotype similarity has proven to reflect the relationship of functionally
related genes. We applied phenotype similarities between various diseases for expanding
molecular connections of cancer-type-specific variants to discover cancer-type-specific
modules. Specifically, cancer-type-specific variants of 7 cancer types from The Cancer
Genome Atlas (TCGA) were analyzed to find phenotype-inferred relationships among the
variants. We find that cancer variants that cause the similar disease phenotypes tend
to be linked as a cluster of biological pathways or functions. Moreover, cancer-type-specific
modules could explain the underlying pathogenicity of specific symptoms which manifest
in particular cancer types. Cancer-type-specific modules and pathways found from phenotype
similarity/dissimilarity based on cancer symptoms improved the discrimination performance
to sort cancer-type-specific variants to accurately predict patient groups. Our method
will be further developed to find genetic biomarkers for the diagnosis or prognosis
of specific cancer types
PK-009
Engineering a stable, symmetric membrane protein scaffold
Amanda Duran1, Jens Meiler1
1Vanderbilt University-Department of Chemistry
Computational protein engineering has the potential to contribute to various fields
including drug design, protein therapeutics, and materials science. Protein-ligand
interface design and the construction of large, stable proteins rely on stable scaffolds.
Symmetry is a great tool for protein stability both in protein engineering and nature.
Several membrane protein structures exhibit pseudo-symmetry and are proposed to be
the result of gene duplication, fusion and diversification events originating from
a monomeric gene. Aquaporins (AQP) are a class of membrane proteins that exhibits
a two-fold inverted pseudo-symmetry. The Escherichia coli AQP Glycerol facilitator
protein (GlpF) was originally computationally engineered to be perfectly symmetric
in sequence and presumably in structure. The symmetric gene was assembled, cloned,
and expressed. However, after facing many challenges experimentally, the computational
study has been expanded to 13 AQPs of known structure for a more extensive symmetric
backbone search. MAMMOTH structural alignment was used to align the structures to
their inverted counterparts. Cutpoints were calculated based on α-Carbon distance.
Finally, the Rosetta Protein Modeling Software Suite was used to refine and energetically
minimize the symmetric backbones. From over 1500 generated symmetric backbones, 20
candidates were chosen for experimental verification. These studies are ongoing.Currently,
the symmetric backbone models have scored to be more stable than the wild-type proteins.
Experimental verification of these symmetric backbones will provide valuable information
for the current state of membrane protein modeling and design using computational
methods.
PL-001
Intrinsically disordered proteins drive heritable transformations of biological traits
Daniel Jarosz1, James Byers1, Sohini Chakrabortee2, Sandra Jones3, Amelia Chang2,
David Garcia1
1Stanford University, 2Whitehead Institute for Biomedical Research, 3Rockefeller University
The transmission of information from one generation to the next generally occurs via
nucleic acids. The only known protein-based molecular memories are prions, which drive
heritable biological traits based upon self-templating changes in protein conformation.
These protein-based genetic elements have previously been identified systematically,
but at least three do not share the sequence biases or structural characteristics
that have informed such studies. Here we employed a comprehensive library of yeast
proteins to examine the breadth of protein-based inheritance. Transient overexpression
of more than forty proteins created new traits that were heritable and beneficial.
Some shared properties of known prions, but most employed distinct genetic and biochemical
mechanisms to act as elements of inheritance. Traits with these characteristics were
common in wild yeast strains and could also be elicited using orthologous mammalian
proteins. The inducing proteins were strikingly enriched in intrinsically disordered
sequences that have been widely conserved across evolution. Intrinsically disordered
proteins are associated with human disease and with dosage sensitivity in yeast, flies
and worms. Our results suggest another widespread role for such intrinsically disordered
sequences: induction of heritable epigenetic switches that transform phenotypic landscapes
and drive adaptation to stressful environments.
PL-002
Prediction of binding affinity in protein complexes: contacts do matters
Anna Vangone1, Alexandre MJJ Bonvin 1
1Computational Structural Biology group, Bijvoet Center for Biomolecular Research
Almost all critical functions in cells rely on specific protein-protein interactions.
Understanding these is therefore crucial in the investigation of biological systems.
Despite all past efforts, we still lack a thorough understanding of the energetics
of association of proteins. Here, we introduce a new and simple approach to predict
binding affinity based on functional and structural features of the biological system,
namely the network of interfacial contacts. We assess its performance against a protein-protein
binding affinity benchmark and show that both experimental methods used for affinity
measurements and conformational changes have a strong impact on prediction accuracy.
Using a subset of complexes with reliable experimental binding affinities and combining
our contacts- and contact types-based model with recent observations on the role of
the non-interacting surface in protein-protein interactions, we reach a high prediction
accuracy for such a diverse dataset outperforming all other tested methods.
PL-003
Free radical oxidation – a new method for obtaining stable protein coatings on magnetic
nanoparticles
Anna Bychkova1, Alexandra Vladimirova1, Mariya Nezhivaya1, Tatiana Danilova1, Pavel
Pronkin1, Maria Gorobets1, Alexander Tatikolov1, Vyacheslav Misin1, Mark Rosenfeld1
1N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences
Magnetically targeted nanosystems (MTNSs) are now considered to be applicable in different
areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay,
cell and molecular separation, a smart delivery of drugs to target cells. Proteins
are promising materials for creation of coatings on magnetic nanoparticles (MNPs)
due to their biocompatibility, an ability to protect magnetic cores from influence
of biological liquids and prevent agglomeration of MTNSs in dispersion, their possible
functional activity as therapeutic products and biovectors. The creation of stable
protein coatings with retention of native properties of molecules is still an important
biomedical problem because of disadvantages of the commonly used methods such as formation
of a polydisperse ensemble of particles, nonselective linking of proteins leading
to cross-linking of macromolecules in solution, and desorption of coatings.
A novel method in obtaining stable single-layer coatings assembled from protein molecules
on the surface of magnetite nanoparticles has been developed. It is based on protein
liability to free radical modification, leading to the formation of intermolecular
covalent cross links. Free radicals are locally generated on the surface of nanoparticles
via the Fenton reaction thereby proteins adsorbed on the surface are subjected to
the cross-linking. O-phenylenediamine was used for detection of free radical generation
initiated by nanoparticles. The proteins drastically differing in their structure
and properties, namely, serum albumin, thrombin and immunoglobulin G were selected
for creating the protein coatings. The properties of the obtained coatings and their
stability have been studied with the help of dynamic light scattering (DLS), UV/Vis
spectrophotometry, antibody-antigen test and the method of spectral-fluorescent probes.
Albumin molecules in MNPs coatings have been shown to retain their capability of binding
with a dye and be conformationally stable. The dye 3,3'-di-(γ-sulfopropyl)−5,5'–diphenyl-9-ethiloxacarbocyanine-betaine
interacting with albumin with a growth of fluorescence and with partial cis–trans
conversion of the dye has been used.
It has been proven that coatings composed of protein macromolecules are 1) stable,
2) formed around individual nanoparticles and 3) have several nanometers in thickness.
The free radical linking of thrombin and immunoglobulin G on the surface of nanoparticles
has been shown to almost completely keep native properties of the protein molecules.
The free radical linking method reveals new possibilities for design of single-layer
multiprotein polyfunctional coatings on the surfaces of all the nano-, micro- and
macroobjects containing metals of variable valence (for example, Fe, Cu, Cr).
The spectral-fluorescent investigation was supported by the Russian Foundation for
Basic Research, project nos. 13-03-00863 and 14-03-31196mol_a.
PL-004
Regulation of neuronal SNAREs by accessory proteins
Shrutee Jakhanwal1, Reinhard Jahn1
1Department of Neurobiology, Max Planck Institute of Biophysical Chemistry
Regulation of Neuronal SNAREs by accessory proteins 1Shrutee Jakhanwal and 1Reinhard
Jahn 1Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Fassberg,
Goettingen, Germany-37075. Synaptic vesicle exocytosis lies at the heart of the process
of neurotransmitter release. And, the family of proteins that is central to the process
of synaptic vesicle exocytosis is the family of SNARE proteins. There are three kind
of neuronal SNARE proteins namely Syntaxin, SNAP25 and Synaptobrevin. These three
SNARE proteins interact through their SNARE-motifs to form a highly stable four-helix
bundle, which in turn, pulls two membranes together to mediate fusion. Years of work
in this field have established that the four-helix bundle is critical for the membrane
fusion to occur. However, the process of regulation of SNARE-mediated fusion remains
very poorly understood. The major regulatory proteins involved in the process are
Munc 18, Munc 13, Synaptotagmin and Complexin. The major aim of my project is to obtain
a closer look at the regulation process of SNARE-mediated fusion by focusing on the
interaction between the SNARE proteins and the regulatory proteins. To achieve this
objective, I express and purify the different proteins involved in the process of
SNARE-mediated fusion and thereafter subject them to appropriate biochemical characterization.
In order to assess the role of the purified proteins in the process of fusion, I reconstitute
them into liposomes and perform in-vitro lipid-mixing assays. These assays are based
on Fòrster Resonance Energy Transfer (FRET). Based on the discretion of assessing
the protein-protein or protein-lipid interactions, either the proteins or the lipids
can be fluorescently labeled. Also, the lipid compositions can be varied in order
to assess the effect of lipid on the function of the respective protein. Fluorescence-based
anisotropy measurements can also provide information about the degree of freedom of
a protein, indirectly providing information about the kinetics of a reaction. Employing
these techniques, I observe that Munc 18-1 leads to displacement of Syntaxin from
a complex of Syntaxin and SNAP25. Also, a complex of Syntaxin and Munc 18 is resistant
to the action of the AAA-ATPase, NSF and its co-factor αSNAP, implicating this complex
as a strong candidate for acting as the starting point for the process of neurotransmitter
release. Munc 18 also appears to enhance lipid-mixing by interacting with the SNARE-complex.
Further investigations on the same lines can provide very useful insights into the
process and can help us unravel the secrets that underlie the beauty of the exquisitely
regulated process of neurotransmitter release.
PL-005
Binding of thymidine nucleotides to a viral thymidine monophosphate kinase
Aldo A. Arvizu-Flores1, Eduardo Guevara-Hernández2, Enrique F. Velazquez-Contreras1,
Francisco J. Castillo-Yañez1, Luis G. Brieba3, Rogerio R. Sotelo-Mundo2
1Universidad de Sonora, Departamento de Ciencias Químico Biológicas, 2Centro de Investigación
en Alimentación y Desarrollo, A.C., 3Laboratorio Nacional de Genómica para la Biodiversidad
Nucleotide phosphorylation is a key step towards DNA replication during viral infections
since suitable levels of nucleotide triphosphates pool are required for DNA synthesis.
Deoxythymidine triphosphate (dTTP) is produced either by de novo or salvage pathways,
being the enzyme thymidine monophosphate kinase TMK) at the junction of both pathways.
The TMK from the white spot syndrome virus (WSSV) represents a major target for drug
design directed to shrimp quaculture applications in many countries. This enzyme catalyzes
the ATP-dependent phosphorylation of deoxythymidine monophosphate (dTMP) to yield
deoxythymidine diphosphate (dTDP). In this work, we used isothermal titration calorimetry
and molecular docking to assess the binding thermodynamics for hymidine nucleotides,
such as dT, dTMP, dTDP and the nucleosidic analog of phosphorylated stavudine (d4TMP),
to the TMK from WSSV. Dissociation onstants (Kd) for dTMP and dTPDP were 3.2 and 6.2
μM, respectively. Both ligands showed similar binding energetics, with a subtle difference
of 1.5 kcal/mol in the enthalpic component. Interestingly, the Kd for d4TMP was 3.5
μM, whereas for dT was 11.6 μM. For all thymidine nucleotides, the Kd values are almost
dentical to that of dTMP, despite of being different in one hydroxyl or phosphate
group. These results suggest that nucleoside analogues like d4TMP, could be considered
as a feasible treatment strategy for the WSSV disease in farmed shrimp.
PL-006
A cold-adapted trypsin in sardine fish (Sardinops sagax caerulea): molecular modeling
and recombinant expression
Aldo A. Arvizu-Flores1, Manuel I. Carretas-Valdez2, Francisco J. Castillo-Yañez1,
Karina D. Garcia-Orozco3, Carmen A. Contreras-Vergara3, Rogerio R. Sotelo-Mundo3,
Maria A. Islas-Osuna1,3
1Departamento de Ciencias Químico Biológicas, Universidad de Sonora, 2Departamento
de Investigación y Posgrado en Alimentos, Universidad de Sonora, 3Centro de Investigación
en Alimentación y Desarrollo
THEME: Biochemistry There is great interest in the evolution and activities of fish
trypsins, since they appear to have evolved into different families. The cDNA for
trypsin III from the Monterey sardine (Sardinops sagax caerula) was obtained and its
deduced amino acid sequence matched its identity with a purified protease from the
fish by mass spectrometry analysis. Molecular modeling of sardine trypsin III compared
to other homologs showed a typical trypsin fold with all the cognate components for
catalysis, and specific amino acid distribution that are possible factors that explain
the cold adaptation. From phylogenetic analysis, sardine trypsin III belongs to the
novel Y family, which is proposed to have evolved for cold adaptation. The obtained
recombinant trypsin III showed a low catalytic efficiency, but it remained active
at cold temperatures, similar to other cold-adapted trypsins. The cold-adaptation
of sardine trypsin III opens a wide range of biotechnological applications for this
protease and is also interesting from the serine protease structure-function relationship
point of view.
PL-007
Fungicidal mechanism of scolopendin 2, a cationic antimicrobial peptide from centipede
Heejeong Lee1, Dong Gun Lee1
1Kyungpook National University
Scolopendin 2, AGLQFPVGRIGRLLRK, is a 16-mer peptide derived from the centipede Scolopendra
subspinipes mutilans. To investigate its property against fungal and bacterial pathogens,
antimicrobial tests were performed. We observed that this peptide exhibited antimicrobial
activity in a salt-dependent manner and showed no hemolysis. The circular dichroism
(CD) analysis observed that α-helical structure properties. We determined the mechanism(s)
of action using flow cytometry and investigated the release of potassium. The results
showed that the microbial membrane in Escherichia coli O157 and Candida albicans was
permeabilized with loss of potassium ions. Additionally, the bis-(1,3-dibutylbarbituric
acid) trimethine oxonol [DiBAC4(3)] and 3,3’-dipropylthiacarbocyanine iodide [DiSC3(5)]
assay showed membrane depolarization. Using calcein-encapsulating giant unilamellar
vesicles (GUVs) and FITC-dextran containing large unilamellar vesicles (LUVs), scolopendin
2 disrupted the cell membrane and the damage size is between 4.8 to 5.0 nm against
composition of microbial plasma membrane of E. coli and C. albicans. Thus, we demonstrated
that a cationic antimicrobial peptide, scolopendin 2, possesses broad-spectrum antimicrobial
effects that formed pore on the cell membrane.
PL-008
Structural and functional investigation of the far C-terminal domain (CTD) of the
bifunctional enzyme traI using NMR Spectroscopy
Krishna Chaitanya Bhattiprolu, Evelyne Schrank, Klaus Zangger
Protein Structural Biology Structural and functional investigation of the far C-terminal
domain (CTD) of the bifunctional enzyme TraI using NMR Spectroscopy B.Krishna Chaitanya,
Evelyne Schrank and Klaus Zangger Institute of Chemistry/Organic and Bioorganic Chemistry
University of Graz, Austria Corresponding author email ID: krishna.bhattiprolu@uni-graz.at
Bacterial conjugation is a complex process for the horizontal transfer of single stranded
DNA from one cell to another. This mechanism also leads, for example, to the spread
of antibiotic resistance genes and virulence factors among bacterial species. Multi-protein
complexes formed at the origin of transfer (oriT) region of DNA and at the cytoplasmic
membrane of the bacterial cell, initiate this process. Inside the membrane, the relaxosome
identifies the single strand for transfer in a plasmid DNA, relaxes and unwinds it,
whereas the transferosome is involved in pilus formation (Type IV secretion system)
and transferring the gene through the cytoplasmic membrane. These events take place
in the donor bacterial cell along with several other auxiliary proteins [1] The bifunctional
enzyme TraI of plasmid R1 plays a crucial role in the relaxosome activity, as it contains
both a relaxase and helicase domain. To exert its functions on DNA, TraI works in
close co-ordination with other relaxosome proteins like TraY, TraM and the integration
host factor. TraI is a 1756 residual protein and contains 3 major domains: N-terminal
relaxase domain, a central helicase domain and a C terminal domain (CTD). The structure
of the C-terminal domain until residue 1629 has been solved by crystallography, while
the structure and function of the remaining ∼130 residues remained undetermined [2].
There are SAXS models and crystallographic structures for different parts of TraI
and also for the full length protein. All of them miss the information about this
particular domain. This region is of interest, particularly because it is an intrinsically
disordered (ID) region and ID regions in most of the proteins are involved in regulatory
functions. Previous mating pair experiments have strong evidences which show that
the translocation frequency of the conjugative DNA decreases drastically (from 6.5
x 10-3 to 2 x 10-3 colonies) upon deletion of this ∼130 residues domain from the full
length TraI. We are investigating the structure and function of this very C-terminal
end of TraI using NMR spectroscopy. For the backbone assignment we used slice-selectively
homonuclear broadband decoupled spectra along with standard experiments. Three-bond
scalar coupling constants were obtained through real-time J-upscaling experiments.
With the backbone assignments, we have the first hand evidence which shows that his
domain is for the most part intrinsically disordered, but contains short α-helical
regions. Structural development, interaction studies to find the binding partner and
transition of disorder to order orientation of this domain will be further investigated
in this project.
References:
[1] Redzej, Adam et al. “Structure of a Translocation Signal Domain Mediating Conjugative
Transfer by Type IV Secretion Systems.” Mol. Microbiol.89 (2) (2013): 324–333.
[2] Guogas, Laura M. et al. “A Novel Fold in the Trai Relaxase-Helicase C-Terminal
Domain Is Essential for Conjugative DNA Transfer.” J. Mol. Biol. 386(2) (2009): 554–568.
PL-009
Sodium chloride induced aggregation of monoclonal antibodies at low ph: prevention
by additives
Fabian Bickel1, 2, Hans Kiefer1
1Institute of Applied Biotechnology, Biberach University of Applied Sciences, 2International
Graduate School in Molecular Medicine Ulm, Ulm University
Protein Aggregation Aggregation is a known phenomenon in downstream processing of
monoclonal antibodies (mAbs) in connection with certain stress factors like shear
forces, low pH values and high salt concentrations. Here we investigated a model system
where mAb aggregation is induced by increasing the ionic strength (NaCl) at low pH.
The aggregation depends both on protein and sodium chloride concentration. With Nanoparticle
Tracking Analysis (NTA) and Micro Flow Imaging (MFI) the aggregation formation was
further characterized. Aggregation can be partially reverted by lowering the ionic
strength as determined by soluble monomer concentration measurement using SE-HPLC:
Parts of insoluble aggregates could be solubilized as soluble aggregates, dimers or
even monomers. A quasi equilibrium is formed in between the subtypes. The whole aggregation
process was examined by FTIR and CD-Spectroscopy to identify structural changes of
the mAb. Screen of protective additives: The effect of osmolyte additives on aggregation
kinetics and final aggregate concentration is investigated, revealing protective effects
in both cases. In a screen with more than 200 compounds not only the aggregation propensity
was studied but also structural changes. The Aggregation Index (quantity for colloidal
stability) and the melting point (quantity for conformational stability) measured
by differential scanning fluorimetry were determined. The used MTP format screen has
potential for buffer optimization and formulation development.
PL-011
Conformational flexibility of CD81 cellular receptor head-subdomain – implications
on Hepatitis C binding modes
Eva S. Cunha1, Pedro Sfriso2, Adriana Rojas1, Adam Hospital2, Modesto Orozco2, Nicola
Abrescia1
1Structural Biology Unit, CIC bioGUNE, 2Institute for Research in Biomedicine (IRB
Barcelona)
Structural Biology and Protein Dynamics Tetraspanin CD81 has a broad range of cellular
functions, such as integrin association forming tetraspanin-enriched domains, synapse
formation between B and T cells, cell adhesion, motility, invasion and signalling.
Furthermore, CD81 is one of the four receptors involved in the cell entry of Hepatitis
C virus (HCV) and therefore infection onset, one of the major causes for chronic liver
disease resulting in cirrhosis and hepatocarcinoma. Human CD81 Large-Extracellular-Loop
(hCD81LEL) is composed of a “stalk” and a “head” subdomain; with the latter interacting
with HCV-E2 glycoprotein. We present four novel hCD81LEL crystal forms. Analysis of
the fourteen independent observed hCD81LEL high-resolution X-ray structures suggests
that the dynamism of the hCD81LEL head-subdomain is an inherent molecular property,
an observation supported also by Molecular Dynamics (MD) studies. We classify the
conformations in three distinct clusters (closed, intermediate and open), which are
seen both in the crystal structures and in the molecular dynamics simulations. The
MD simulations also show that conformational variability is modulated by pH changes,
with distinct probability for each cluster at acidic and neutral pH. Furthermore,
in silico docking of the recent E2core structure with three of the major types of
hCD81LEL head-subdomain clusters highlights hydrophobic interactions as the major
forces in the E2core: hCD81LEL recognition mechanism. We propose that the flexibility
of the hCD81LEL is exploited by HCV at different stages of cell entry from virus attachment
to internalization and fusion with the endosomal membrane. Our results provide important
insights on the basic mechanism governing HCV binding to hCD81, and can help structure-based
drug design of entry-inhibitors of HCV.
PL-012
Allophycocyanin of gracilaria chilensis: from gene to function
Jorge Dagnino-Leone1, José Martinez-Oyanedel1, Marta Bunster-Balocchi1
1Universidad de Concepción
THEME: Structure-Function relationship of proteins
The phycobilisomes (PBS) are auxiliary photosynthetic complexes that allow cyanobacteria
and red algae to enhance the energy uptake in the range of 490-680 nm. In Gracilaria
chilensis, an eukaryotic red algae, PBS is composed of Phycoerythrin (PE), Phycocyanin
(PC) and Allophycocyanin (APC); these proteins possess chromophores which capture
energy and then transfers it to photosytems. PBPs are oligomers of a αβ heterodimer;
it oligomerizes into a trimer (αβ)3, this trimer has discoidal shape and it is associated
in hexamers (αβ)6, several of this hexamers forms cylinder-like structures. PBS has
2 components: antennas and core. The antennas are composed of PE and PC, whose function
is to capture energy between 490-570 and 590-625 nm respectively and transfer it to
the core. The core is formed by APC, which can absorb energy in the 620-650 nm range.
APC emission allows transferring energy to the photosystems with high efficiency.
PBS is also composed by linker proteins which allow the correct assembly of PBS and
possibly regulate the energy transfer. The main goal in our group is to build an atomic
model of the Gracilaria chilensis phycobilisome. We have solved the crystal structure
of PE and PC and created an antenna model. At present we are working in APC and the
chromophorilated linker proteins. The objective of the present work is to create a
model of the core of Gracilaria chilensis; to achieve these we have used molecular
biology, biochemistry and bioinformatics techniques. We designed oligonucleotides
primers for the four allophycocyanin subunits genes and for the globular domain of
the apcE linker. These primers were used in PCR experiments to obtain the genes sequences.
The sequences were translated to a aminoacid sequences and used to build a 3D model
for APC subunits and trimers using the software Modeller. On the other hand we purified
and analyzed the spectroscopic properties of APC from Gracilaria chilensis using absorption
and fluorescence spectroscopy. We also determined APC oligomerization state using
Gel filtration. Molecular docking using the CLUSPRO server was performed to obtain
a hexamer and APC cylinder models. Based on electron micrographs obtained by our lab
a tri-cylindric core model was built. All the models were submitted to a molecular
dynamics using GROMACS software. Finally we determine possible energy transfer pathways
in the core model applying the extended Forster equation, spectroscopic data from
literature and the transition dipole moments of each of the chromophores present in
the core. As conclusion of this work we built the first atomic model of Gracilaria
chilensis phycobilisome core and propose energy transfers pathways inside the core
in the context of a phycobilisome.
PL-013
Novel practical strategies to access artificial metalloenzymes
Marco Filice1, Jose Miguel Palomo1
1Departamento de Biocatálisis, Instituto de Catálisis, CSIC
Protein Chemistry and Engineering Since the first report, the design of artificial
metalloenzymes has rapidly been converted into an important topic in biological and
inorganic chemistry due to their potential applications in synthetic chemistry, nanoscience
and biotechnology. The combination of a catalytically active organometallic moiety
with a macromolecular host has permitted the creation of biohybrids, a new kind of
heterogeneous catalytic entities combining the attractive features of both homogeneous
and enzymatic systems. Presenting our most recent achievements in this research area,
here we describe two novel powerful and promising approaches focusing the practical
synthesis and large scale production of heterogeneous artificial metalloenzymes showing
chimeric activity. The first strategy is based on the in situ synthesis of noble metal
nanoparticles and their supramolecular assembly with a microbial lipase from Candida
antarctica (fraction B) finally creating an ultra-active organometallic-enzyme heterogeneous
nanobiohybrid. In the second approach, combining different protein engineering protocols
(molecular biology, orienting immobilization, solid-phase bioorganic modification
and bioinformatic tools), an orthogonal solid-phase strategy creating novel unnatural
catalytic sites was designed and optimized. The application of such a strategy onto
the structure of the lipase from Geobacillus thermocatelunatus permitted the generation
of a heterogeneous artificial metallolipase with chimeric activity. As proof-of-concept,
the combinatorial library of generated artificial metalloenzymes obtained by both
strategies was successfully assessed in a set of different synthetic reactions (selective
C-C bond formation as Suzuki, Heck or Diels-Alder reactions) and also combining both
activities (metallic and enzymatic) in cascade processes such as dynamic kinetic resolution
of amines or production of arylamines. The obtained results were excellent in all
cases. Extending this strategy to other enzymes, proteins and catalytic metals, we
envisage the creation of a combinatorial library of programmable artificial enzymes
useful for a wide set of applications (i.e. fine organic and medicinal chemistry,
bioremediation or biomedicine).
PL-014
Proteomic examination of the yeast nuclear pore complex dynamics
Zhanna Hakhverdyan1, Kelly Molloy2, Brian Chait2, Michael Rout1
1Laboratory of Cellular and Structural Biology, 2Laboratory of Mass Spectrometry and
Gaseous Ion Chemistry
Protein turnover and exchange Nuclear pore complexes (NPCs) are proteinaceous assemblies
situated in nuclear envelopes of eukaryotic cells. The main function of the NPC is
the selective transport of macromolecules. NPCs also partake in other functions, such
as nuclear organization and gene regulation. The core scaffold of the NPC is thought
to be a stable structure, while the peripheral components exchange at various rates.
However, these phenomena have not been elucidated in detail. The recent findings that
yeast daughter cells get a higher proportion of the old NPCs and the core scaffold
hardly turns over raise the possibility that the exchange of the peripheral nucleoporins
can be a repair mechanism. Yeast provides a useful organism for the interrogation
of nucleoporin exchange, as it performs closed mitosis; hence the only mixing of NPC
constituents is due to exchange. We have developed a panel of genetic tools providing
for conditional induction and repression of nucleoporins. By combining these switches
with stable isotope metabolic labeling and affinity capture, cross linking coupled
to mass spectrometry, we are able to distinguish between pre-existing and newly synthesized
proteins and quantify their relative amounts in the NPC. Our preliminary findings
are in agreement with results obtained in other organisms: the core scaffold of the
NPC (inner ring, outer ring) appears to be stable, however does exchange slowly over
time, while peripheral components exchange faster. By looking at the exchange rates
of yeast nucleoporins we hope to gain insight into the NPC biology of actively dividing
eukaryotic cells.
PL-015
Active site clustering identifies functional families of the peroxiredoxin superfamily
Angela Harper1, Janelle Leuthaeuser2, Patricia Babbitt2, Jacquelyn Fetrow3
1Department of Physics, Wake Forest University, 2Department of Molecular Genetics
and Genomics,Wake Forest University, 3Departments of Physics and Computer Science,
Wake Forest University
Bioinformatics Understanding the relationships between proteins is vital to increasing
our knowledge of the protein universe. While there are large databases of sequence
information, the massive data influx over the past decade has prevented adequate classification
of proteins at the molecular function level. However, it has been previously suggested
that a protein’s active site information may correlate with these known molecular
functional differences; thus, active site profiling was developed to use residues
around the active site of a protein to relate proteins. Subsequently the Deacon Active
Site Profiler (DASP) was developed to create these active site profiles and search
them in a database, such as GenBank, in order to find proteins with similar active
site environments. By using DASP to computationally cluster proteins based on the
similarity of their active site profiles, the Peroxiredoxin (Prx) superfamily was
analyzed through active site similarity methods. The residues from the active site
of each Prx structure were extracted and clustered, and these profiles were iteratively
searched in GenBank through a Multi-level Iterative Sequence Searching Technique (MISST).
The Prx superfamily has been studied by experts, allowing the results of these searches
to be compared to a well-annotated group of proteins. While previous sequence based
evolutionary methods have been unable to identify functional differences between some
subgroups of the Prxs, notably the AhpC-Prx1 and Prx6 subgroups, MISST discretely
separates these subgroups. Classifying Prx proteins into functionally relevant groups
using computational active site similarity methods lays the foundation for an automated
process for identifying protein functional groups beyond the Prx superfamily.
PL-016
Synthesis and conformational studies of glycoprotein N homolog of bovine herpesvirus
1 (BHV-1) by using CD, NMR and molecular modelling
Natalia Karska1, Andrea D. Lipińska2, Małgorzata Graul2, Franciszek Kasprzykowski1,
Emilia Sikorska1, Igor Zhukov3, Magdalena J. Ślusarz1, Sylwia Rodziewicz-Motowidło1
1Faculty of Chemistry, University of Gdansk, 2Intercollegiate Faculty of Biotechnology,
University of Gdansk, 3Nano Bio Medical Centre University of Poznan
UL49.5 protein (gN homolog) is a key player in the immune evasion strategies of several
varicelloviruses, including bovine herpesvirus-1. During viral infection UL49.5 exerts
dual activity: it serves as a chaperone for viral glycoprotein M and, in its gM-unbound
form, acts as an inhibitor constraining the transporter associated with antigen processing
(TAP). The UL49.5/gM complex formation is required for the maturation and proper trafficking
of both viral proteins. In the absence of gM, UL49.5 blocks transport of antigenic
peptides by TAP and their MHC I-restricted presentation. The molecular mechanism of
UL49.5 activity still remains elusive. In order to investigate the structural requirements
for biological function UL49.5 study was conducted using CD, NMR and Molecular Dynamics
methods. The data obtained with the use of high purity synthetic peptides encompassing
UL49.5 confirmed the presence of an alpha-helix structure, formed preferentially in
the presence of dodecylphosphocholine (DPC) micelles as a membrane-like environment.
In order to determine the three-dimensional structure of UL49.5 protein in the present
work its NMR solution structure in the presence of membrane-like environment was performed.
The NMR data were used as a set of restraints for a simulated annealing protocol that
generated 3Dstructures of the peptides in DPC micelles [3]. In the next step, the
calculation of spatial structure and “assembling” of the whole protein from the obtained
peptide structures were performed by using molecular dynamics of the protein in the
fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) [4]. The
obtained structural model may contribute to identification of UL49.5 active sites
and elucidation of its mode of action.
[1] Verweij M.C., Lipińska A.D., (2011) Molecular Immunology 48, 2038- 2051
[2] Verweij M.C., Koppers-Lalic D, (2008) The Journal of Immunology 181: 4894-4907.
[3] Marassi, F.M., Opella, S.J., Determining, I., National, B., Protein, L., Bank,
D., 1998. NMR structural studies of membrane proteins. Curr. Opin. Struct. Biol. 8,
640–8
[4] Ślusarz, M.J., Gieldoń, A., Ślusarz, R., Trojnar, J., Meadows, R., Ciarkowski,
J., 2005. QSAR Comb. Sci. 24, 603–610. doi:10.1002
Acknowledgments:
Polish National Centre for Research and Development - grant number 178479
PL-017
Functional and mechanistic studies of dysferlin, an essential protein in cell membrane
repair
Colin Johnson1, Sara Codding1
1Oregon State University
Membrane proteins Resealing of tears in the sarcolemma of myofibers is a necessary
step in the repair of muscle tissue. Defects in this repair process are responsible
for muscular dystrophy and cardiomyopathy. The repair pathway is triggered by the
influx of calcium through lesions in the membrane, which result in membrane fusion
and patching of the wound. Recently dysferlin has been identified as a calcium binding
protein essential for sarcolemma repair, as well as other SNARE mediated exocytotic
events including cytokine and acid sphingomyelinase secretion. In this presentation
we demonstrate a direct interaction between dysferlin and the SNARE proteins syntaxin
4 and SNAP-23. In addition, FRET and in vitro reconstituted lipid mixing assays indicate
that dysferlin accelerates SNARE heterodimer formation and SNARE mediated lipid mixing
in a calcium sensitive manner. Our results suggest a model whereby dysferlin acts
as a calcium sensing SNARE effector for exocytosis and membrane fusion.
PL-018
Exploring the therapeutic potential of a peptide derived from a poxviral immune evasion
protein: NMR determination of the solution structure of VIPER and its inactive mutant
Jiyoon Kim1, Dylan Lawless1, Manuel Ruether2, Andrew Bowie1, Kenneth H. Mok1,3,
1Trinity College Dublin, Trinity Biomedical Sciences Institute (TBSI), 2Trinity College
Dublin, School of Chemistry, 3Trinity College Dublin, Centre for Research on Adaptive
Nanostructure/devices
Toll-like receptors (TLRs) have a role in viral detection leading to cytokine and
IFN induction, and as such they are targeted by viruses for immune evasion. The poxviral
protein A46 has been identified to inhibit TLR signaling by interacting with TIR domain-containing
proteins of the receptor complex to collectively inhibit all TLR adaptor proteins
that positively regulate transcription-factor activation (1). One 11 aa peptide (KYSFKLILAEY)
termed VIPER (Viral Inhibitory Peptide of TLR4) was reported to retain the inhibitory
properties of full length A46 against TLR4 signaling. A 9R homopolymer delivery sequence
at the C-terminus provided delivery of the peptide into cells. Structural comparisons
are presented between 9R-VIPER, which is active in preventing TLR4-dependent cytokine
induction in cell culture, and a mutant that exhibited loss of function (9R-VIPER
L6A,E10A), through solution NMR spectroscopy. We find that despite a relatively minor
sequence difference, the loss of hydrophobicity as well as negative electrostatic
interactions result in subtle but potentially significant differences in the region
of the peptide proposed to interface with TLR4.
Reference:
(1) Stack J, Bowie AG, “Poxviral protein A46 antagonizes Toll-like receptor 4 signaling
by targeting BB loop motifs in Toll-IL-1 receptor adaptor proteins to disrupt receptor:adaptor
interactions “J Biol Chem 287: 22672-82, 2012.
PL-019
Active site profile-based protein clustering is an efficient, accurate method to define
protein functional groups
Janelle Leuthaeuser1, Angela Harper2, Gabrielle Shea2, Patricia Babbitt3, Jacquelyn
Fetrow1,2
1Wake Forest University, 2Wake Forest University, 3University of California San Francisco
Protein Function Prediction The elucidation of protein molecular function lags far
behind the rate of high-throughput sequencing technology; thus, it is essential to
develop accurate and efficient computational methods to define functional relationships.
Protein clustering based on sequence similarity has emerged as a simple, high-throughput
method for defining protein relationships, but sequence-based techniques often inaccurately
define molecular function details. Active site profiling (ASP) was previously developed
to identify and compare molecular details of protein functional sites. Protein similarity
networks were created using both active site similarity and sequence similarity for
four manually curated superfamilies, and results demonstrate that ASP-based clustering
identifies detailed functional relationships more accurately than sequence-based clustering.
Building on this, two iterative pipelines were developed using active site profiling
and profile-based searches to cluster protein superfamilies into functional groups.
First, the Two Level Iterative clustering Process (TuLIP) utilizes active site profiling
and iterative PDB searches to divisively cluster protein structures into groups that
share functional site features. Across eight superfamilies, TuLIP clusters exhibit
high correlation with expert functional annotations. Subsequently, the Multi-level
Iterative Sequence Searching Technique (MISST) utilizes iterative profile-based GenBank
searches to identify protein sequences that belong in each TuLIP group. The results
indicate that these ASP-based methods accurately and efficiently identify functionally
relevant groups through a process that can be applied systematically and on a large-scale.
Moreover, the approach can be applied more quickly than detailed manual curation,
suggesting its value in guiding annotation efforts.
PL-020
Insertion of the hydrophobic C-terminal domain of apoptotic BH3-only proteins into
biological membranes
Ismael Mingarro1, Vicente Andreu-Fernández2, Manuel Bañó-Polo1, Maria J. García-Murria1,
Mar Orzáez2
1Dept. Biochemistry and Molecular Biology. University of Valencia, 2Lab of Peptide
and Protein Chemistry. Centro de Investigación Príncipe Felipe
Membrane Proteins Changes in the equilibrium between pro-survival and pro-apoptotic
members of the B-cell lymphoma-2 (Bcl-2) protein family at the mitochondrial outer
membrane (MOM) induce structural changes that committed cells to apoptosis. Bcl-2
homology-3 (BH3)-only proteins participate in this process activating pro-apoptotic
effectors and promoting permeabilization of the MOM. The membrane association of BH3-only
proteins is a controversial issue due to the lack of a canonical carboxyl-terminal
(C-terminal) transmembrane (TM) domain. We used an in vitro transcription/translation
system to study the insertion capacity of these hydrophobic C-terminal regions of
the BH3-members Bik, Bim, Noxa, Puma and Bmf into microsomal membranes, and an Escherichia
coli complementation assay to validate our results in bacterial cells. Furthermore,
we have fused these hydrophobic regions to GFP to investigate the subcellular sorting.
These results will allow further refinement in the elaboration of the Bcl-2 protein-protein
and protein-membrane interactome network.
PL-021
A computational investigation of tight junctions
Alexis Peña1, Flaviyan Jerome Irudayanathan1, Shikha Nangia1
1Syracuse University, Dept. of Biomedical and Chemical Engineering
Computational Modeling, Biostatistics, Biomedical and Chemical Engineering Tight junctions
(TJ) are vital intracellular barriers that are responsible for regulating paracellular
transport. Claudins, a family of small transmembrane proteins with approximately 27
members, are an integral part of the TJ strands. Tight junctions provide molecular-level
protection and prevent infection and toxins from entering the body; in the same sense
TJs allow nutrients and vital solutes to pass through. Claudins are associated with
various diseases including metastatic cancer as well as an entry point for many viruses.
Despite their importance and abundance in all cell membranes and their ubiquitous
nature, the exact 3-D structure of Claudins has remained elusive to traditional X-ray
crystallographic and NMR studies. In this investigation, a computational approach
was used to determine the Claudin structure of claudin 1-10. Homology modeling, molecular
dynamic simulations, and reverse mapping were employed to predict the protein structures
with relative accuracy. Understanding structure of claudin proteins and its interaction
at the molecular level can lead to effective drug delivery technology.
PL-022
Determination of optimal conditions for an isothermal titration calorimetry essay
to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (Sardinops
sagax caerulea)
Idania Emedith Quintero Reyes1, Francisco Javier Castillo Yáñez1, Enrique fernando
Velázquez Contreras1, Rocío Sugich Miranda1, David Octavio Corona Martínez1, Aldo
Alejandro Arvizu Flores1, Ivet Cervantes Domínguez1
1Universidad de Sonora
Protein Kinetics Determination of Optimal Conditions for an Isothermal Titration Calorimetry
Essay to Obtain Kinetic Parameters of Trypsin I from Pyloric Caeca of Monterey Sardine
(Sardinops sagax caerulea) Trypsin is the most studied alkaline protease and it´s
very common to found isoforms from this protein as the case for Monterey sardine (Sardinops
sagax caerulea); as it shows an expression of trypsin I and trypsin III according
to the cDNA characterization. Trypsin I was determine to be a cold adapted enzyme
as it shows a higher catalytic efficiency (kcat/KM) than the mesophilic counterparts.
The kinetic parameters were obtained by spectrophotometric essays, which are not fallible
for all the enzymes because native, recombinant or mutant enzyme activity could be
below the detection limit of the assay, opaque or turbid solutions interfere with
spectrophotometric detection, etc. Alternative tools as the isothermal titration calorimetry
(ITC) can measure enzyme kinetics using thermal power generated by the enzymatic conversion
of substrate to product; were the rate of reaction is directly proportional to thermal
power. The objective of this study was to stablish the optimum conditions to obtain
kinetic parameters of Trypsin I from pyloric caeca of Monterey sardine using ITC.
To reach the objective Trypsin I was purified from viscera of Monterey sardine using
molecular exclusion and affinity chromatography obtaining a yield of 1.1 mg/mL. At
20°C kcat and KM of Tryipsin I form Monterey sardine were 14.6 s-1 and 1.4 µM respectively.
At 15°C were 13.6 s-1 and 4 µM (kcat and KM) and at 4°C kcat was 0.454 s-1 and KM
0.52 µM. The kinetic parameters obtained by spectrophotometric assay at 25°C were
kcat and KM 436 s-1 and 1.8 µM respectively. At 20°C the kcat was 409.7 s-1 and KM
1.8 µM and at 15°C kcat 288 s-1 and KM 3µM. Comparing the values obtained for kcat
with the spectrophotometric essay were higher 29 fold than those obtained by ITC and
the values in KM were similar by both methods. Even though the differences in kcat,
we can reassert the psychrophilic behavior of trypsin I as the catalytic efficiency
is higher by both methodologies. In the understanding that the kinetic behavior of
enzymes is important to not only understanding biochemical pathways and catalytic
mechanisms but is again a fruitful area for drug discovery and development; so the
ITC provides a universal approach to determining the kinetic behavior of enzymes and
can yield in a single experiment a complete set of kinetic parameters for an enzyme-catalyzed
reaction that can be applied for the different alkaline proteases from pyloric caeca
of Monterey sardine (Sardinops sagax caerulea).
PL-023
Mysterious world of stress-responding sigma factors in Bacillus subtilis
Olga Ramaniuk1
1Institute of Microbiology, Academy of Sciences of The Czech Republic
Protein-DNA interaction Bacterial transcription is mediated by the RNA polymerase
holoenzyme containing sigma factors - essential proteins for the initial step of transcription
that recognize and bind to promoter DNA. The primary sigma factor is essential in
exponential phase of growth while alternative sigma factors are active during transcription
under stress conditions. This project has three main aims. The first aim is to explore
the binding properties of B. subtilis alternative sigma factors; specifically, whether
sigma factors lacking the autoinhibitory domain 1.1 can bind to promoter DNA in the
absence of RNAP. The second aim explores whether RNAP associated with alternative
sigma factors is regulated by the concentration of the initiation nucleoside triphosphate.
The third aim is to define the regulon of Sigma I. In order to achieve our aims, 7
out of 17 alternative sigma factors were successfully purified using affinity chromatography
and ion exchange chromatography. We set up in vitro transcription system with selected
sigma factors and initiated experiments with Sigma I regulon determination. Results
named above and our future findings will help to better understand gene expression
regulation on the level of transcription initiation.
This work was supported by grant No. P305-12-G034 from the Czech Science Foundation.
PL-024
Assessing the costs and benefits of protein aggregation
Natalia Sanchez de Groot1, Marc Torrent Burgas2, Charles N. J. Ravarani1, Salvador
Ventura3, M. Madan Babu1
1MRC Laboratory of Molecular Biology, 2Vall d’Hebrón Research Institute, UAB, 3Inst.
de Biotec. i Biomed. and Dprt. de Bioq. i Biol. Mol., UAB
Protein aggregation and cell fitness Protein aggregation has been associated with
numerous diseases but also with important cellular functions such as epigenetic inheritance.
Here we present a population genetics approach to infer the costs and benefits of
protein aggregation on cell fitness. This information is crucial to understand how
cellular systems tolerate the formation of protein deposits and which factors modulate
this event. Using our experimental system, we measured different protein aggregation
effects (deleterious, neutral or beneficial) within the same genomic background. Single
cell analyses, within the same population, showed stochastic variability in the aggregate’s
size and in its effect on cell fitness. Our data indicates that, in certain conditions,
protein aggregation can enhance population variability and survival expectancy. Overall,
these results suggest that the presence and formation of protein aggregates could
be almost harmless whereas the associated gain and loss of function are critical for
the cell.
PL-025
Revealing the key role of negatively charged residues of heme sensor proteins involved
in Geobacter sulfurreducens’ signal transduction pathways
Marta A. Silva1, Telma C. Santos1, Teresa Catarino2, Carlos A. Salgueiro1
1UCIBIO-Requimte, Departamento de Química, FCT-UNL., 2Instituto de Tecnologia Química
e Biológica, UNL
Signal transduction proteins Bacterial chemotaxis systems sense and regulate the microbe
mobility in response to environmental conditions. Such mechanisms constitute a striking
example of cell motility to gain advantages for cell survival and permit the bacteria
to fill important niches in a diversity of anaerobic environments [1]. Geobacter sulfurreducens
(Gs) is an anaerobic bacterium with a considerable respiratory versatility whose genome
encodes for an unusual family of methyl-accepting chemotaxis proteins (MCP), each
containing at least one heme c-binding motif [2]. These sensor proteins, GSU0582 and
GSU0935, are involved in signal transduction pathways mediated by chemotaxis-like
systems [3]. The thermodynamic and kinetic characterization of the sensors GSU0582
and GSU0935 by visible spectroscopy and stopped-flow techniques, at several pH and
ionic strength values revealed that sensor GSU0935 midpoint reduction potentials are
lower than those of GSU0582 at all pH and ionic strength values and the same were
observed for the reduction rate constants [4]. The origin of the different functional
properties of these closely related sensor domains are rationalized in the structural
terms showing that GSU0935 has two extra negatively charged residues in the vicinity
of the heme group, which have no counterpart in GSU0582: Glu89 and Asp57. Residue
Asp57 is less exposed compared to Glu89 and it was suggested that its carboxylic group
might have a role in the modulation of the heme reduction potential of GSU0935. To
investigate this, both residues were replaced by a positively charged amino acid (lysine)
and by a neutral one (asparagine or glutamine). For the mutants with enough expression,
a functional characterization was carry out, using several spectroscopic techniques,
including UV-visible and CD, together with kinetics and potentiometric measurements.
Significant changes on the reduction potential values are observed when a negative
charge is replaced by a positive one at position 57 or 89. Therefore, the decrease
of the reduction potential in Asp57 and Glu89 mutants reinforces the hypothesis that
the higher reduction potential observed for heme sensor domain GSU0582 is related
with the less negative electrostatic surface around the heme. This work provides,
for the first time, evidence for the co-existence of two similar methyl-accepting
chemotaxis proteins functioning in different working potential ranges. These proteins
are responsible to allow Geobacter sulfurreducens triggering an adequate cellular
response in different anoxic subsurface environments.
Acknowledgments:
This work was supported by project grant PTDC/BBB-BEP/0753/2012 and UID/Multi/04378/2013
from Fundação para a Ciência e a Tecnologia (FCT), Portugal. TCS is recipient of grant
PD/BD/106037/2015 from FCT, respectively.
References:
1. MK Chan. Curr Opin Chem Biol, 5 (2001) 216-222.
2. DR Lovley, JD Coates. Curr Opin Microbiol, 3 (2000) 252-256.
3. PR Pokkuluri, M Pessanha, YY Londer, SJ Wood, NE Duke, R Wilton, T Catarino, CA
Salgueiro, M. Schiffer. J Mol Biol, 377 (2008) 1498-1517.
4. MA Silva, RC Valente, PR Pokkuluri, DL Turner, CA Salgueiro, T Catarino. Biochim
Biophys Acta, 1837 (2014) 920-928.
PL-026
Appearance of stabilizing interactions in the evolution of a dimeric TIM barrel
Mariana Schulte-Sasse1, Nancy O. Pulido Mayoral1, Miguel Costas-Basín2, Enrique García-Hernández3,
Adela Rodríguez-Romero3, D. Alejandro Fernández-Velasco1
1National Autonomous University of Mexico, Faculty of Medicine, 2National Autonomous
University of Mexico, Faculty of Chemistry, 3National Autonomous University of Mexico,
Institute of Chemistry
Molecular Evolution The glycolytic enzyme triosephosphate isomerase (TIM) is an oligomeric
(β/alpha)8 barrel that catalyses the interconversion of D-Glyceraldehyde 3-phosphate
and dihydroxyacetone phosphate in a diffusion-limited reaction. Although each subunit
has its own active site, naturally occurring monomeric TIMs have not been reported;
in fact, monomer association is very tight. TIM topology is well conserved among the
three domains of life. Nevertheless, their folding mechanism and inhibition properties
vary across species. Comparative studies of proteins have proved to be very useful
in understanding the relationship between sequence and physicochemical properties,
however, they lack the capacity to give a more integrative and evolutive correlation.
In order to elucidate how the catalytic properties, the oligomerization state and
the stability of extant TIMs arose, in this work we examined the molecular history
of eukaryotic TIM through ancestral protein reconstruction methods (Maximum Likelihood)
and the subsequent physicochemical characterization of the resurrected enzymes. We
first characterized in detail the protein corresponding to the last common ancestor
of animals and fungi (TIM63). The CD and fluorescence spectra of TIM63 are similar
to those of extant TIMs. Secondary structure is lost in a cooperative transition with
Tm = 68.7°C. The enzyme loses activity upon dilution suggesting that only the dimer
is active. Dilution experiments followed by isothermal titration calorimetry indicate
that dissociation enthalpy is small; moreover the heat capacity change observed is
three times higher than the one predicted for a rigid body dissociation process, suggesting
partial unfolding of the monomers. When compared with extant TIMs, the catalytic efficiency
of TIM63 is reduced 10-fold, whereas binding of PGH, a transition-state analogue,
shows a similar thermodynamic signature. These data indicate that although monomer
association may have been less tight in ancestral TIMs, catalysis has been always
linked to oligomerization. Analysis of the crystal structure of TIM63, obtained at
1.9 Å resolution, suggests that the lack of four salt bridges observed in the interface
of extant TIMs is responsible for the low dimer stability. In order to test this hypothesis
we also studied the stability of four younger reconstructed ancestors that acquired
the salt bridges in two different phylogenetic lineages. We found a correlation between
the appearance of stabilizing interactions in the interface, dimer stability and catalysis;
suggesting that these salt bridges are partially responsible for extant dimer stability
and shed light on the dimeric nature of extant TIMs.
PL-027
Receptor protein-tyrosine phosphatases: dimerization, receptor kinase interaction
and allosteric modulation
Elizabeth Dembicer1, Damien Thevenin1
1Department of Chemistry, Lehigh University
THEME: receptor tyrosine kinase and receptor protein phosphatase signaling Many cell-signaling
events are regulated through reversible tyrosine phosphorylation of proteins, which
is controlled by the counterbalanced actions of two key enzyme families: Protein tyrosine
kinases and protein tyrosine phosphatases. Interestingly, both families include transmembrane
receptor-like enzymes, namely the receptor tyrosine kinases (RTKs) and the receptor-like
PTPs (RPTPs). While the regulation and actions of many RTKs are well characterized,
the mechanisms controlling the enzymatic activity of RPTPs and how they interact with
their substrates remain to be fully explained. Thus, understanding how these receptors
function and interact will give fundamental insights into how tyrosine phosphorylation
is finely tuned in cells, and how it can be modulated. Increasing evidence indicates
that RPTPs, like RTKs, are regulated by homodimerization. However, it appears that
homodimerization inhibits the activity of most RPTPs. Even though the transmembrane
(TM) and the juxtamembrane domains have been proposed to be involved in this process,
there is no clear structure-based proposal for the role of these regions. Moreover,
several RPTPs have been identified as candidate regulators of RTKs. In particular,
the Receptor-type tyrosine-protein phosphatase eta (PTPRJ; also known as DEP1 or CD148)
is capable of attenuating EGFR tyrosine phosphorylation. Physical interactions of
EGFR with PTPRJ at the cell surface have been documented, but the basis for these
interactions is unknown. Here, using a dominant-negative transcriptional activator-based
assay (DN-AraTM), and mutagenesis analysis, we show that: (1) PTPRJ has a strong tendency
to homodimerize, (2) PTPRJ heterodimerizes with EGFR through TM-TM interactions, (3)
these interactions are mediated by specific residues, and can be modulated by the
delivery of peptide binders. This work represents the first structure-function study
of RPTP-RTK interaction, and may not only result in significant progress towards a
better understanding of the basic biology of RPTPs in cancer cells, but also offer
new possibilities for targeting protein tyrosine phosphatases for therapeutic modulation
of EGFR in oncology.
PL-028
Inhibiting EGFR dimerization and signaling through targeted delivery of juxtamembrane
domain peptide mimics using pHLIP
Anastasia Thevenin1, Kelly Burns1, Janessa Guerre-Chaley1, Damien Thevenin1
1Department of Chemistry, Lehigh University
Regulating Receptor Tyrosine Kinase Signaling The elevated phosphorylation of key
regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein
tyrosine kinases activity plays well-established roles in promoting tumorigenesis
and in the high frequency with which resistance arises to existing therapeutic treatment.
For instance, this is the case for the epidermal growth factor receptor (EGFR). Thus,
there is a clear need for novel specific targeting methods to inhibit the activity
of receptor protein tyrosine kinases, such as EGFR, in cancer. EGFR becomes activated
upon ligand binding to the extracellular domain, leading to receptor dimerization.
The juxtamembrane (JM) domain of EGFR is critical for intrinsic tyrosine kinase activity
and receptor dimerization by stabilizing the active conformation of EGRR through the
formation of a antiparallel helical dimer. Therefore, peptides mimicking the JM domain
– if specifically delivered to cancer cells – have the potential to prevent EGFR dimerization,
receptor activation, downstream signaling, and thus to attenuate aberrant EGFR activity
in cancer cells. Here, pHLIP (pH Low Insertion Peptide), a peptide that can selectively
target cancer cells and tumors based solely on their extracellular acidity, is used
to selectively translocate the JM domain of EGFR in cancer cells to prevent EGFR dimerization.
At pH above 7, pHLIP is soluble and unstructured, however, when exposed to lower pH
such as observed in tumors, pHLIP inserts as a transmembrane (TM) alpha-helix, allowing
the direct translocation of cargo molecules into the cytoplasm. Using the dominant
negative AraC-based transcriptional reported assay (DN-AraTM), which assesses JM and
TM domain interactions in cells membranes of E. coli, we show that pHLIP-JM is able
to disrupt EGFR dimer by 50%. Current work is focused on testing the ability of such
pHLIP-JM peptide conjugate to perturb EGFR homodimerization and decrease downstream
signaling through soluble kinases, such as Akt and ERK, in cancer cells.
PL-029
The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity,
transcript fidelity and mitochondrial transcription factor binding
Gilberto Velazquez1, Luis Brieba2, Rui Sousa3
1Universidad de Guadalajara, 2Langebio Cinvestav, 3University of Texas HealthSscience
Center at San Antonio
DNA protein interaction ABSTRACT Single subunit RNA polymerases have evolved two mechanisms
to synthesize long transcripts without falling off a DNA template: binding of nascent
RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a
common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we
characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41)
in complex stability, processivity, and fidelity. We found that deletion and point
mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of
abortive transcripts and the probability that the polymerase will disengage from the
template during the formation of the late initial transcription and elongation complexes.
Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric
template and, unexpectedly, thumb subdomain deletions decrease the binding affinity
for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb
subdomain is part of an extended bindingsurface area involved in binding Mtf1.
PL-030
Design principles of membrane protein structures
Vladimir Yarov-Yarovoy1, Diane Nguyen1
1University of California Davis
Membrane Protein Structure Membrane proteins play key role in cellular signaling and
ion transport. Statistical analysis of expanding database of high-resolution membrane
protein structures in Protein Data Bank (PDB) provides useful information about membrane
protein structure and function. We used RosettaMembrane software (Yarov-Yarovoy V
et al (2006) Proteins) to analyze ∼300 unique alpha helical membrane protein structures
in PDB and derive knowledge based energy function for membrane protein structure prediction,
membrane protein-protein docking, and membrane protein design. The RosettaMembrane
residue environment energy term is based on amino acid propensities in hydrophobic,
interface, and water layers of the membrane and depends on the residue burial state
– from being completely buried within a protein environment to being completely exposed
either to the lipid or water environments. Residue buried state is determined from
the number of residue neighbors within 6 and 10 Å spheres. The RosettaMembrane residue-residue
interaction term is based on the propensities of amino acid pairs to be in close proximity
to each other within hydrophobic, interface, and water layers. Results of our statistical
analysis reveal fine details of favorable and unfavorable environments for all amino
acids types in all membrane layers and residue burial states. We find that large hydrophobic
amino acids are favorable facing the hydrophobic core of the lipid bilayer. Small
amino acids are favorable facing the protein core within the hydrophobic layer of
the membrane. Aromatic or positively charged amino acids and favorable facing the
lipid head groups. Residue-residue interactions are often favored between polar and
charged amino acids and also between some of small and large hydrophobic amino acids
inside of the protein core within the hydrophobic layer of the membrane. These data
will be useful for rational design of novel membrane protein structures and functions.
PL-031
Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine
Ohad Yosefson1, Andrew Nager1, Tania Baker1, Robert Sauer1
1Department of Biology, Massachusetts Institute of Technology
Protein quality control’ or ’Protein degradation’ Hexameric AAA+ protein-remodeling
machines use conserved loops that line the axial pore to apply force to substrates
during the mechanical processes of protein unfolding and translocation. An open question
in the AAA+ field is whether pore loops from different subunits of the hexameric ring
grip the substrate coordinately (all six subunits involved), independently (one subunit
at a time involved), or partially coordinated (two or three subunits at a time). To
answer this question, we studied covalently linked hexamers of the E. coli ClpX unfoldase
bearing different numbers and configurations of wild-type and mutant pore loops and
challenged these variants with protein substrates with a broad range of stabilities.
We find that successful unfolding of increasingly resistant substrates requires the
coordinated action of a greater number of wild-type pore loops. Our results support
a mechanism in which a power stroke initiated in one subunit of the ClpX hexamer results
in the simultaneous movement of all six pore loops, which coordinately grip and apply
force to the substrate.
PL-032
Structure and function of the Toc159 M-domain, and its role in targeting the preprotein
receptor to the chloroplast outer envelope membrane
Matthew Smith1, Shiu-Cheung Lung2, Prem Nichani1, Nicholas Grimberg1, J. Kyle Weston1,
Shane Szalai1, Simon Chuong2
1Deartment of Biology, Wilfrid Laurier University, 2Department of Biology, University
of Waterloo
Chloroplast biogenesis and function rely on the import of thousands of nucleus-encoded
preproteins from the cytosol. Preprotein import is supported by the Toc and Tic (Translocon
at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively
to translocate preproteins across the double-membrane envelope to the chloroplast
interior. Toc159 is one of the preprotein receptors of the Toc complex, is also encoded
in the nucleus and post-translationally targeted to the chloroplast, and is comprised
of 3 distinct domains: 1) the intrinsically disordered N-terminal Acidic (A-) domain;
2) the central GTPase (G-) domain; and 3) the C-terminal Membrane (M-) domain that
anchors the protein to the chloroplast outer membrane (COM) through an unknown mechanism.
The M-domain has no known homologues and does not contain a predicted trans-membrane
domain, but does contain intrinsic chloroplast targeting information at the extreme
C-terminus. The M-domain also contains a predicted β-helix motif, which may be important
for anchoring the protein to the COM. We are interested in characterizing the structure
of the M-domain and determining the nature of its association with the COM, as part
of our larger goal of understanding the role Toc159 plays in protein import into chloroplasts.
We are also interested in defining the precise nature of the targeting information
contained within the extreme C-terminus of Toc159, elucidating the targeting pathway
that is used, and whether other COM proteins use this pathway. We will present our
most recent data on the structure, function and targeting of the Toc159 M-domain.
PL-033
Structural investigation of NlpC/P60 protein acquired by Trichomonas vaginalis through
a lateral gene transfer event
Jully Pinheiro1,2, Augusto Simoes-Barbosa1, David Goldstone2
1Microbiology, School of Biological Sciences, University of Auckland, 2Structural
Biology, School of Biological Sciences, University of Auckland
Trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes
the most common non-viral sexually transmitted disease, with approximately 200 million
cases worldwide annually. Nevertheless, the biochemical processes behind T. vaginalis
infection and its interaction with the vaginal microbiota are still not well defined.
In 2007 the draft genome sequence of Trichomonas vaginalis strain G3 was described,
identifying 60,000 protein-coding genes. Of these, nine genes encode NlpC/P60-like
members. This superfamily is widely represented in the different kingdoms of life
and has diverse enzymatic functions, such as amidases, endopeptidases and acetyltransferases.
Previous studies have shown that members of this superfamily hydrolyze specific peptide
linkages in bacterial cell walls affecting germination, vegetative growth, sporulation
and division or cell lysis/invasion. As a typical eukaryote, the protozoan parasite
T. vaginalis does not have a cell wall itself. Previous studies suggest that the T.
vaginalis NlpC/P60 genes were acquired via lateral gene transfer from bacteria and
must have an important function, possibly controlling the vaginal microbiota and aiding
parasite invasion and infection. To investigate the function of the NlpC/P60 family
of proteins in T. vaginalis we have expressed, purified and crystallized a member
TVAG_119910 and report its three-dimensional structure, determined at 1.5 Å resolution,
by X-ray diffraction. The structure of the protein reveals a typical papain-like fold
resembling peptidoglycan hydrolases from the NlpC/P60 family with a conserved cysteine
and histidine; forming the catalytic residues. The protein contains two bacterial
SH3 domains at the N-terminus. This domain acts as a general binding domain and is
likely to aid the interaction of the NlpC/P60 domain with substrate components. Combined
with biochemical and enzymatic characterization, the structure of this NlpC/P60 protein
will help to elucidate the molecular origin of its hydrolase activity and to decipher
their putative role in the parasite infection.
PL-034
Novel DNA polymerases from Red Sea brine-pools: new potential polymerases for PCR
application
Masateru Takahashi1, Etsuko Kimura1, Mohamed Salem1, Ulrich Stingl1, Samir Hamdan1
1King Abdullah University of Science and Technology
Protein Biotechnology The polymerase chain reaction (PCR) is a key tool in medical
and biological research. The most common PCR reaction relies on the thermal cycling
method that consists of repeated cycles of heating and cooling steps for DNA melting
and extension by the DNA polymerase, respectively. The introduction of new DNA polymerases
to the market is a major area of development that tremendously helped in improving
the performance and quality of PCR. Nonetheless, PCR still requires optimization of
salt and metal ion concentrations leaving a room in the market for introducing new
DNA polymerases that are robuster in their salt and metal ion concentration dependence.
In this study, we will present the characterization of a novel archaeal DNA polymerase
from the Red Sea brine-pool (termed BR3) and demonstrate how its enzymatic activity
reflects on every aspects of the environment of the brine-pool – high tolerance to
concentrations and types of salts and metal ions including utilization of Zn2+ ions
in its active site. These results suggest that the brine-pool microorganisms are likely
to contain novel chemical pathways to deal with its exterior harsh conditions. We
will further show the mechanism of BR3 polymerase how it was adjusted to be active
in harsh condition.
PL-035
Structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid
protein as an antiviral target
Ming-Hon Hou1, Shing-Yen Lin1, Chia-Ling Liu1, Yu-Ming Chang2, Jincun Zhao3, Stanley
Perlman3
1Institute of Genomics and Bioinformatics, National Chung Hsing University., 2Institute
of Biological Chemistry, Academia Sinica., 3Department of Microbiology, The University
of Iowa
Drug Discovery Coronaviruses (CoVs) cause numerous diseases, including Middle East
respiratory syndrome and severe acute respiratory syndrome, generating significant
health-related and economic consequences. CoVs encode the nucleocapsid (N) protein,
a major structural protein that plays multiple roles in the virus replication cycle
and forms a ribonucleoprotein complex with the viral RNA through the N protein’s N-terminal
domain (N-NTD). Using human CoV-OC43 (HCoV-OC43) as a model for CoV, we present the
3D structure of HCoV-OC43 N-NTD complexed with ribonucleoside 5’-monophosphates to
identify a distinct ribonucleotide-binding pocket. By targeting this pocket, we identified
and developed a new coronavirus N protein inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)(N,N-dimethylamino)acetamide
hydrochloride (PJ34), using virtual screening; this inhibitor reduced the N protein’s
RNA-binding affinity and hindered viral replication. We also determined the crystal
structure of the N-NTD-PJ34 complex. On the basis of these findings, we propose guidelines
for developing new N protein-based antiviral agents that target CoVs.
PL-036
Thermal and structural stability of ß-Glucosidases GH1
Maira Artischeff Frutuoso1
1Departamento de Bioquímica do Instituto de Química da Universidade de São Paulo
Enzymology We compared the stability of thermophilic β-glucosidases GH1 to mesophilic
ones in the presence of denaturants as urea and high temperature by following the
transitions between the native and unfolded states by tryptophan fluorescence, enzymatic
activity and differential scanning fluorimetry (DSF). The bacterial β-glucosidases
(bglA) and (bglB) of the mesophile Paenibacillus polimyxa and β-glucosidase (bglThm)
of the thermophile Thermotoga maritima were expressed as recombinant proteins in NovaBlue
(DE3) and purified by affinity chromatography (Ni-NTA resin). These recombinant enzymes
have very similar folding type structure (β/α)8 barrel, as shown in crystal structures
and exhibited a characteristic peak between 330 and 340 nm in the tryptophan fluorescence
spectra, indicating that those proteins are folded. Circular dichroism analysis in
the far-UV region (190 nm to 240 nm) also showed typical spectra of folded proteins
with secondary structure composition of 47% of α-helix and 13% of β-sheets for bglA,
61% of α-helix and 2.5% of β-sheets for bglB and 30% of α-helix and 20% of β-sheets
for bglThm. The average degree of accessibility to the exposed tryptophan residues
in the native enzyme to increasing concentrations of the acrylamide suppressor (Stern-Volmer
constant - KSV) is greater to bglA (9.49), but similar to bglB (3.17) and bglThm (3.84).
The thermal stability determined by DSF was higher for bglB (Tm 43.8ºC) than for bglA
(Tm 35.2ºC). The bglThm was stable at 47°C and remained stable for up to 4 h at 80°C.
In addition the thermal inactivation kinetics at 47°C evaluated by the relative remaining
activity showed that bglA denaturation (kinactivation of 1.9 s-1) is faster than bglB
(kinactivation of 31.3 s-1). On the other site, bglThm inactivation at 95°C was a
two-step process, which exhibited an initial fast step (kinactivation of 2.9 s −1)
followed by a slow step (kinactivation of 0.2 s-1). The chemical denaturation by urea
followed using tryptophan fluorescence showed a transition (c50) at 7.9 ± 0.2 mol•L-1
for bglA and 7.1 ± 0.2 mol•L-1 for bglB, the bglThm was stable at 9M of urea (showing
95% of native state). Moreover the ’m’ parameter, which represents the denaturant
effect on the protein stability, is 669 cal•mol-1 for bglA and 860 cal•mol-1 for bglB.
In conclusion, bglB showed intermediate stability between bglA and bglThm. Supported
by FAPESP and CAPES.
PL-037
Computational modeling of INI1/SMARCB1 and novel insights into its interaction with
HIV-1 Integrase
Savita Bhutoria1, Sheeba Mathew2, Menachem Spira2, Xuhong Wu2, Kalpana Ganjam2, Seetharama
Acharya1
1Department of Hematology, Albert Einstein College of Medicine, 2Department of Genetics,
Albert Einstein College of Medicine
Protein structure modeling, protein- protein interaction Computational modeling of
INI1/SMARCB1 and novel insights into its interaction with HIV-1 Integrase Savita Bhutoria1,
Sheeba Mathew2, Menachem Spira2, Xuhong Wu2, Ganjam V Kalpana2* and Seetharama A Acharya1*
Departments of 1Hematology and 2Genetics, Albert Einstein College of Medicine, Bronx,
New York. *Equal corresponding authors The INI1/SMARCB1 gene encodes a component of
the SWI/SNF ATP-dependent, chromatin-remodeling complex. INI1/SMARCB1 is present in
all mammalian SWI/SNF complexes. It was isolated as an interacting protein for HIV-1
Integrase (IN) and subsequently demonstrated to be associated with cMYC, the carboxyl-terminal
SET domains of ALL-1 and EBNA (Epsteain Bar Virus, nuclear antigen)1. INI1/SMARCB1
has no known structural homologues, and its amino-acid sequence yields little insight
into its function. A detailed understanding of structure-function relationships is
hampered by the lack of structural information for INI1. Computational methods that
model protein/peptide structures with sufficient accuracy to facilitate functional
studies have had notable successes. We carried out combination of sequence analysis
ab initio structure modeling and dynamics studies of Integrase Binding Domain of INI1
and found it to be similar to that of Phospholipase A2 Activating Protein, PLAA. Structural
similarity with this distant protein suggests divergent evolution of the two proteins.
The modeled structure sheds light on various protein-protein interactions of INI1.
By integrating the experimental studies about the binding, we have shown through docking,
how a fragment of INI1 binds to the HIV-1 IN. Molecular docking and experimental studies
indicated that two proteins bind tightly through charged/polar residues surrounding
a hydrophobic cleft. These studies provide first modeled structure of INI1/SMARCB1
or any component of the SWI/SNF complex, and provide structural basis for IN-INI1
interactions. This molecular interpretation of the intermolecular interactions is
expected to facilitate design of inhibitors as novel class of anti-HIV-1 therapeutic
agents.
References:
Morozov A, Yung E, Kalpana GV (1998) Structure-function analysis of Integrase Interactor
1/hSNF5L1 reveals differential properties of two repeat motifs present in the highly
conserved region. ProcNatlAcadSciUSA 95: 1120–1125.
Maillot B, Lévy N, Eiler S, Crucifix C, Granger F, et al. (2013) Structural and Functional
Role of INI1 and LEDGF in the HIV-1 Preintegration Complex. PLoS ONE 8(4): e60734.
PL-038
Structural determinants for human RNase 6 antimicrobial mechanism of action
Javier Arranz Trullén1, Guillem Prats-Ejarque1, Jose Antonio Blanco1, Marcel Albacar1,
Diego Velazquez1, David Púlido2, Mohammed Moussaoui1, Ester Boix1
1Department of Biochemistry and Molecular Biology, Biosciences Faculty, UAB, 2Department
of Life Sciences, Imperial College
Host Defence & Immunity Structural determinants for human RNase 6 antimicrobial mechanism
of action. The RNase A superfamily is a vertebrate specific family that includes eight
functional members in humans. Together with their catalytic activity towards RNA substrates,
other biological properties have been reported and evolution studies suggest an ancestral
host-defence function in vertebrates. Indeed, genetic studies confirmed a rapid molecular
evolution within the family, a distinctive trait for host defence proteins exposed
to a changing pathogen environment. Previous studies from our laboratory characterized
the wide spectra antimicrobial activity of two highly cationic human RNases: the eosinophil
RNase 3 and the skin derived RNase 7 (Boix et al., 2012). However, the family photo
still remained incomplete. In the present study we have explored the structural determinants
required for human RNase 6 mechanism of action. RNase 6 is a secretion protein expressed
in innate cell types. Its induced secretion at the urinary tract during infection
suggests a physiological protective role (Becknell et al., 2014)(Pulido et al., 2013).
We present here the characterization of the RNase catalytic activity together with
its membrane binding mode and bactericidal properties. Our results show that the protein
displays a high antimicrobial activity against both Gram negative and Gram positive
species together with a cell agglutinating ability. By applied site-directed mutagenesis
we have spotted the protein residues contributing to the protein distinctive features.
Becknell, B., Eichler, T. E., Beceiro, S., Li, B., Easterling, R. S., Carpenter, A.
R., … Spencer, J. D. (2014). Ribonucleases 6 and 7 have antimicrobial function in
the human and murine urinary tract. Kidney International, 87, 151–161.
Boix, E., Salazar, V. a., Torrent, M., Pulido, D., Nogués, M. V., & Moussaoui, M.
(2012). Structural determinants of the eosinophil cationic protein antimicrobial activity.
Biological Chemistry, 393(August), 801–815.
Pulido, D., Torrent, M., Andreu, D., Nogues, M. V., & Boix, E. (2013). Two human host
defense ribonucleases against mycobacteria, the eosinophil cationic protein (RNase
3) and RNase 7. Antimicrobial Agents and Chemotherapy, 57(RNase 3), 3797–3805.
PL-039
Covalent structure of single-stranded fibrinogen and fibrin oligomers cross-linked
by fxiiia. The influence of free radical oxidation
Anna Bychkova1, Vera Leonova1, Alexander Shchegolikhin1, Marina Biryukova1, Elizaveta
Kostanova1, Mark Rosenfeld1
1N. M. Emanuel Institute Of Biochemical Physics, Russian Academy Of Sciences
Protein structure and function Native fibrinogen is a key blood plasma protein whose
main function is to maintain hemostasis by virtue of producing the cross-linked fibrin
clots under the effect of thrombin and fibrin-stabilizing factor (FXIIIa). FXIIIa-mediated
isopeptide γ–γ bonds are known to be produced between γ polypeptide chains of adjacent
fibrinogen or fibrin molecules. But there are apparently conflicting ideas regarding
the orientation of γ–γ bonds. In this study several peculiarities of self-assembly
of fibrin(ogen) and induced oxidation of the proteins have been studied with the aid
of elastic and dynamic light scattering, UV-, FTIR- and Raman spectroscopy methods.
In the presence of FXIIIa both the non-oxidized and oxidized fibrinogen molecules
has been shown to bind to each other in the “end-to-end” fashion to form the flexible
covalently cross-linked fibrinogen homopolymers. To identify the orientation of γ–γ
bonds in fibrin protofibrils a novel approach based on self-assembly of soluble cross-linked
fibrin protofibrils and their dissociation in the urea solution of moderate concentrations
has been applied. The results of elastic and dynamic light scattering coupled with
analytical ultracentrifugation indicated the protofibrils to exhibit an ability to
dissociate under increasing urea concentration to yield single-stranded structures
entirely brought about by γ–γ bonds. The results of this study provide an evidence
to support the model of the longitudinal γ–γ bonds that form between the γ chains
end-to-end within the same strand of a protofibril. Since fibrinogen is known to be
sensitive to ROS the mechanisms of fibrinogen and fibrin self-assembly under induced
oxidation have been investigated. In both cases the polypeptide chains of the oxidized
fibrin(ogen) proved to be involved in the enzymatic cross-linking more readily than
those of unaffected molecules. The enhancing role of the D:D interaction under oxidation
could be considered as an compensatory mechanism in the assembly of fibrin when the
D:E interaction is impaired. The experimental data on fibrinogen and fibrin oxidation
acquired in the present study, being combined with our earlier findings, make it reasonable
to suppose that the spatial structure of fibrinogen could be evolutionarily adapted
to some ROS actions detrimental to the protein function.
The study was supported by the RFBR, Research Projects 14-04-31897mol_a and 15-04-08188a.
PL-040
Structural and thermodynamic analysis of co-stimulation receptor CD28 phosphopeptide
interactions with Grb2, Gads, and PI3-kinese SH2 domains
Satomi Inaba1, Nobutaka Numoto2, Hisayuki Morii3, Teikichi Ikura2, Ryo Abe4, Nobutoshi
Ito2, Masayuki Oda1
1Graduate School of Life and Environmental Sciences, Kyoto Prefectural University,
2Medical Research Institute, Tokyo Medical and Dental University (TMDU), 3National
Institute of Advanced Industrial Science and Technology, 4Research Institute for Biomedical
Sciences, Tokyo University of Science
In addition to the signaling produced by the binding of antigen-major histocompatibility
complex to T-cell receptors, co-stimulatory signals from other receptor-ligand interactions
are required for full activation of T-cells. The CD28 receptor on the T-cell surface
has been well characterized, and the binding of ligand to CD28 is critical for producing
co-stimulatory signals. CD28 has no enzymatic activity and its cytoplasmic region
consists of 41 amino acids that contain the sequence YMNM, in which the tyrosine residue
is phosphorylated by kinase. The phosphorylated sequence, pYMNM, is recognized by
Src homology 2 (SH2) adaptor proteins, such as growth factor receptor binding protein
2 (Grb2), Grb2-related adaptor downstream (Gads), and the phosphatidylinositol 3-kinase
(PI3-kinase) regulatory subunit, p85. The consensus sequence for the binding of Grb2
SH2 and Gads SH2 is pYXNX, and that of p85 N-terminus SH2 (nSH2) and C-terminus SH2
(cSH2) is pYXXM. We reported the high-resolution crystal structure of Grb2 SH2 in
complex with the CD28 phosphopeptide [Higo et al., PLOS ONE 8, e74482, 2013], and
recently determined those of Gads SH2, p85 nSH2, and p85 cSH2. These data along with
the results of binding thermodynamics analyzed using isothermal titration calorimetry,
helped to elucidate the molecular recognition mechanisms of CD28 by adaptor proteins.
The SH2 proteins were over-expressed in Escherichia coli, and were purified using
affinity and gel-filtration chromatography. The CD28 phosphopeptides, 8-residue (OctP)
and 12-residue (DdcP02), were synthesized using the solid-phase supported technique,
and were purified using reversed-phase chromatography. The crystals were obtained
by the hanging-drop vapor diffusion method. X-ray diffraction data were collected
at synchrotron radiation facilities, and the structures were determined by the molecular
replacement method. The models of Grb2 SH2, Gads SH2, p85 nSH2, and p85 cSH2 in complex
with OctP were refined at 1.35, 1.2, 1.0, and 1.1 Å resolutions, respectively. The
crystal structures showed that the phosphotyrosine phosphate moiety directly interacted
with the side-chain of arginine in SH2, which is common in all complex structures.
In the Grb2 SH2 and Gads SH2 complexes, the side-chain of asparagine at the pY+2 position
forms a pair of hydrogen bonds with the main-chain amide and carbonyl groups of lysine
in SH2. Alternatively, in the p85 nSH2 and cSH2 complexes, the side-chain of methionine
at the pY+3 position is located in hydrophobic pockets of nSH2 and cSH2, in which
the hydrophobic interactions of cSH2 would be stronger than those of nSH2. This idea
is supported by the observed binding thermodynamics. The binding affinity of cSH2
to DdcP02, because of a favorable enthalpy change, is about 10-fold higher than that
of nSH2. The binding affinity of Grb2 SH2 to DdcP02 is similar to that of Gads SH2
to DdcP02, and is about 10-fold lower than that of nSH2 to DdcP02. These results indicate
that the contribution of hydrophobic interactions of nSH2 and cSH2 at the pY+3 position
are stronger than those of hydrogen bonds of Grb2 SH2 and Gads SH2 at the pY+2 position.
PL-041
Novel kinetochore protein complex from silkworm holocentric chromosomes
Takahiro Kusakabe1, Hiroaki Mon1, JaeMan Lee1
1Kyushu University Graduate School
The kinetochore, which consists of centromere DNA and a multilayered protein complex,
plays important roles in chromosome organization and segregation. Interactions between
chromosomes and spindle microtubules allow chromosomes to congress to the middle of
the cell, and to segregate the sister chromatids into daughter cells in mitosis, which
is followed cytokinesis. In contrast to monocentric chromosomes, in which the centromere
is normally present at a single region on each chromosome, the holocentric chromosomes
have centromeric activity along the entire length of the chromosome. It has been known
that the silkworm, Bombyx mori, has holocentric chromosomes since 1970s, none of silkworm
kinetochore proteins, however, have been identified so far. Here we report the identification
of a novel set of genes for outer kinetochore proteins in silkworm by using bioinformatics
and RNA interference-based screening. Under the hypothesis that depletion of essential
kinetochore genes causes cell cycle arrest in mitosis, we performed RNAi in the silkworm
cell line, BmN4-SID1, targeting a set of candidate genes. Knockdown of five genes
caused significant cell cycle arrest at the G2/M phase. We also found that these five
proteins make a complex, and that all of them are localized along the chromosome arms,
indicating that the silkworm kinetochore extends along the chromosome.
PL-042
Inactivation of βine aldehyde dehydrogenase from spinach by its physiological substrate
βine aldehyde
Rosario A. Muñoz-Clares1, Andrés Zárate-Romero1, Dario S. Murillo-Melo1, Carlos Mújica-Jiménez1,
Carmina Montiel1
1Facultad de Química, Universidad Nacional Autónoma de México
To contend with osmotic stress caused by drought, salinity, or low temperatures some
plants synthesize the osmoprotectant glycine βine (GB) from βine aldehyde (BAL). The
last step—the irreversible NAD+-dependent oxidation of BAL—is catalyzed by ALDH10
enzymes that exhibit βine aldehyde dehydrogenase (BADH) activity. We here report that
the Spinacia oleracea BADH (SoBADH) is reversibly inactivated by BAL in the absence
of NAD+ in a time- and concentration-dependent mode to approximately 50% of the original
activity. Inactivation kinetics are consistent with a partial reversible, two-steps
mechanism that involves the formation of an active non-covalent enzyme•BAL complex
before formation the inactive enzyme-BAL complex. Crystallographic evidence indicates
that in the enzyme previously inactivated by BAL the aldehyde forms a thiohemiacetal
with the nonessential Cys450 (SoBADH numbering) located at the aldehyde-entrance tunnel,
thus totally blocking the access to the catalytic cysteine. Accordingly, BAL does
not inactivate the C450S SoBADH mutant. Two crystal structures of the inactivating
enzyme-BAL complex showed that the trimethylammonium group of BAL is inside the active-site
aromatic box, as in the productive way of binding. This explains why the inactivation
of the A441I mutant—where the binding of the trimethylammonium group is hindered—requires
non-physiologically high BAL concentrations, while the A441C mutant—where the binding
is allowed—is inactivated similarly to the wild-type enzyme. Cys-450 is conserved
in most plant ALDH10 enzymes of known sequence, and in all of them with proven or
predicted BADH activity. Inactivation by BAL appears therefore to be a common feature
of plants BADHs. This short-term regulation may be of great physiological importance
since the irreversibility of the BADH-catalyzed reaction would unbalance the NAD+/NADH
ratio if the aldehyde concentrations are high, the NAD+ concentrations low and the
reaction is not slowed down. Plants BADHs are prone to this situation since they work
under osmotic stress conditions, when high BAL concentrations are required for the
synthesis of high levels of the osmoprotectant GB. The partial nature of the regulatory
mechanism that we are reporting will contribute to prevent both NAD+ exhaustion and
accumulation of the toxic BAL. To the best of our knowledge, this is the first report
of a novel reversible covalent modification of an ALDH enzyme involving its own substrate.
Supported by UNAM (PAPIIT IN217814) and Consejo Nacional de Ciencia y Tecnología (CONACYT
167122) grants to R.A.M.-C.
PL-044
Study of denaturation of proteins by surfactant using the taylor dispersion analysis
and dynamic light scattering
Anna Lewandrowska1, Aldona Jelińska1, Agnieszka Wiśniewska1, Robert Hołyst1
1Institute of Physical Chemistry Polish Academy of Sciences
The protein-surfactant systems are commonly used in biological, pharmaceutical and
cosmetic applications. Ionic surfactants are said to cause unfolding of proteins and
in consequence losing their biological function. Therefore, knowledge of the structure
of protein-surfactant complexes is important to understand the mechanism of denaturation.
In these studies we employed the Taylor dispersion analysis and dynamic light scattering
to study denaturation process of a few proteins under the influence of sodium dodecyl
sulfate (SDS). We carried out series of measurements at constant protein concentration
and varying SDS concentration. The structural changes were analyzed based on the diffusion
coefficients of the complexes which were formed at different surfactant concentration.
We observed that diffusion coefficient for these proteins was decreasing with increasing
concentration of the surfactant at concentrations below the critical micelle concentration
(CMC). The results obtained using the Taylor dispersion analysis are well correlated
with those obtained using dynamic light scattering.
Acknowledgement:
This research was supported by the National Science Center Grant Opus 4 (UMO-2012/07/B/ST4/01400).
1. Lewandrowska, A.; Majcher, A.; Ochab-Marcinek, A.; Tabaka, M.; Holyst, R., Anal.
Chem. 85 (2013)
2. Majcher, A., Lewandrowska, A.; Herold, F.; Stefanowicz, J.; Słowiński, T.; Mazurek,
A.P.; Wieczorek, S.A.; Hołyst, R., Anal. Chim. Acta, 855 (2015)
PL-046
Paraoxonase 1 (Pon1) regulates water homeostasis by controlling the expression of
Fxr and Aqp2 proteins in mice
Marianna Wieloch1,2, Hieronim Jakubowski1,2,3
1Institute of Bioorganic Chemistry, 2Department of Biochemistry and Biotechnology,
University of Life Sciences, 3Dep. of Microbiology Biochemistry & Molecular Genetics,Rutgers-New
Jersey Medical
Paraoxonase 1 (Pon1) regulates water homeostasis by controlling the expression of
Fxr and Aqp2 proteins in mice The kidney is responsible for maintenance of water and
sodium homeostasis, which is controlled by a complex balance of water intake, renal
perfusion, glomerular filtration and tubular reabsorption of solutes, and reabsorption
of water from the renal collecting ducts. Urine volume depends on aquaporin (Aqp)
water channels located in epithelial cells of renal tubules. Aqp2 expression is regulated
by the bile acid receptor Fxr, a transcription factor also known to regulate lipid
and glucose metabolism (Zhang X et al. Farnesoid X receptor (FXR) gene deficiency
impairs urine concentration in mice. Proc Natl Acad Sci U S A 2014;111:2277-82). Inactivation
of the Fxr gene reduces Aqp2 expression and impairs urine concentrating ability, which
leads to a polyuria or urine dilution phenotype. We have previously found that Pon1-/-
mice exhibit a polyuria phenotype and produce twice as much 24-h urine as their wild
type Pon1+/+ littermates (Borowczyk K et al. Metabolism and neurotoxicity of homocysteine
thiolactone in mice: evidence for a protective role of paraoxonase 1. J Alzheimer’s
Dis 2012;30:225-31). Pon1 is expressed in many organs, including the kidney, circulates
in the blood attached to high-density lipoprotein (HDL), participates in homocysteine
(Hcy) metabolism by hydrolyzing Hcy-thiolactone, and contributes to atheroprotective
function of HDL by reducing oxidative stress and protein damage by N-homocysteinylation
(Perła-Kaján J, Jakubowski H. Paraoxonase 1 and homocysteine metabolism. Amino Acids
2012;43:1405-1417). The purpose of the present work was to test a hypothesis that
Pon1 maintains water homeostasis and prevents polyuria by controlling the expression
of Fxr and Aqp2 proteins. Towards this end we quantified Fxr and Aqp2 proteins in
kidneys of Pon1-/- and Pon1+/+ mice by Western blotting using anti-Fxr and anti-Aqp2
antibodies. We found that expression of both FXR and AQP2 was significantly reduced
(2-fold) in kidneys of Pon1-/- mice (n=4) relative to Pon1+/+ animals (n=4). In conclusion,
these findings demonstrate that Pon1/HDL play a critical role in controlling water
homeostasis by controlling the expression of Fxr and its target gene Aqp2.
Supported in part by NCN grants 2011/02/A/NZ1/00010, 2012/07/B/NZ7/01178, 2013/09/B/NZ5/02794,
2013/11/B/NZ1/00091.
PL-047
Development and application of novel non-Ewald methods for calculating electrostatic
interactions in molecular simulations
Ikuo Fukuda1, Narutoshi Kamiya1, Han Wang2, Kota Kasahara1, Haruki Nakamura1
1Institute for Protein Research, Osaka University, 2Freie Universitaet Berlin
To understand the structure, function, and dynamics of protein systems in a microscopic
description, molecular simulation is an important tool. The most time-consuming part
of molecular simulation is the calculation of long-range interactions of the particles.
In particular, appropriate treatment of the electrostatic interaction is critical,
since the simple truncation cannot be used due to the slow decay of the Coulombic
function. Thus, it is highly demanded to calculate the electrostatic interactions
with high accuracy and low computational cost. For this purpose we have developed
the Zero-multipole (ZM) summation method [1]. In this method the artificial periodic
boundary conditions are not necessary and the Fourier part evaluations are not needed,
in contrast to the conventional Ewald-based methods. Instead, a pairwise function
that is suitably redefined from the Coulombic function is used with a cutoff scheme.
The underling physical idea is simple: (a) in a biological system, a particle conformation
for which the electrostatic interactions are well cancelled is more stable than other
conformations [2]; (b) since such well-cancelled conformations are essentially physical,
we should clip a subset of such a conformation out of the conformation within an ad-hoc
given cutoff sphere and calculate the interactions only from this subset. This idea
is realized by a rigid mathematical consideration that leads to the deformation of
the Coulombic function. The efficiency of the ZM method has been validated in applications
to fundamental systems, such as ionic systems [3] and bulk water [4], and heterogeneous
bimolecular systems, including DNA [5], membrane protein [6], motor protein [7], and
DNA-protein complex [8]. In the presentation, we will provide the theory and the numerical
results of our method and show its efficiencies in detail. We will also discuss how
the treatment of the electrostatic calculations seriously affects the simulation results
of protein systems.
[1] I. Fukuda, J. Chem. Phys. 139, 174107 (2013); Fukuda et al., ibid. 140,194307
(2014).
[2] I. Fukuda and H. Nakamura, Biophys. Rev. 4, 161 (2012).
[3] I. Fukuda, et al. J. Chem. Phys. 134, 164107 (2011).
[4] I. Fukuda, et al. J. Chem. Phys. 137, 054314 (2012).
[5] T. Arakawa et al., PLoS One 8, e76606 (2013).
[6] N. Kamiya, et al., Chem. Phys. Lett. 568-569, 26 (2013); T. Mashimo et al., J.
Chem. Theory Comput. 9, 5599 (2013).
[7] Y. Nishikawa, et al., J. Mol. Biol. 426, 3232 (2014).
[8] K. Kasahara, et al., PLoS ONE 9, e112419 (2014).
PL-048
Isolation and characterisation of the zearalenone degrading hydrolase ZenA
Sebastian Fruhauf1, Michaela Thamhesl1, Patricia Fajtl1, Verena Klingenbrunner1, Elisavet
Kunz-Vekiru2, Gerhard Adam3, Gerd Schatzmayr1, Wulf-Dieter Moll1
1Biomin Research Center, 2Christian Doppler Laboratory for Mycotoxin Metabolism (IFA-Tulln),
3IAGZ, University of Natural Resources and Life Sciences
Zearalenone is a mycotoxin produced by Fusarium graminearum and related Fusarium species.
F. graminearum is a powerful plant pathogen and infects major crop plants around the
world. Acute toxicity of zearalenone is low, but due to its structural similarity
to β-estradiol it has binding affinity to the estrogen receptor, which results in
interference with hormonal balance. Typical effects seen in animals include symptoms
like hyperestrogenism and reproductive disorders (reduced fertility, reduced litter
size or swelling of uterus and vulva). To reduce the risk for human and animal health
posed by the ingestion of contaminated food or feed different decontamination strategies
have been studied, including biotransformation. Today many microorganisms are known
to degrade zearalenone, but for most of them the degradation pathway and formed metabolites
remained unknown, hence it is unknown if this degradation also means detoxification.
Only for the fungal strains Trichosporon mycotoxinivorans and Gliocladium roseum ZEN
degradation has been studied in detail and loss of estrogenicity of reaction products
has been confirmed. We screened for, and isolated zearalenone degrading bacteria from
soil samples. The most promising new bacterial isolate was taxonomically assigned
to the species Rhodococcus erythropolis and designated PFA D8-1. The zearalenone catabolism
pathway of PFA D8-1 was found to be identical as known from G. roseum. The primary
reaction product, hydrolysed zearalenone, has so far only been postulated in G. roseum.
We prepared hydrolysed zearalenone by preparative HPLC and showed loss of estrogenicity
in assays with the breast cancer cell line MCF7 and the estrogen reporter yeast strain
YZHB817. A genomic library was prepared and screened in zearalenone degradation deficient
R. erythropolis PR4. The gene encoding zearalenone hydrolase was found and named zenA.
The hydrolase was identified as member of the α/β-hydrolase family and named ZenA.
It was cloned, recombinantly expressed in E. coli and purified by 6 x His-tag mediated
immobilised metal affinity chromatography. Activity of His-tagged and untagged enzyme
ZenA was compared in cleared lysate and ZenA was purified for enzyme characterisation.
The influence of pH and temperature on enzyme activity and stability was evaluated
and kinetic parameters were determined.
PL-049
A new biding site for snake venom C-type lectins?
Maria Cristina Nonato Costa1, Ricardo Augusto Pereira de Pádua1, Marco Aurelio Sartim1,
Suely Vilela Sampaio1
1University of São Paulo, FCFRP
C-type lectins are proteins that bind different glycan molecules by interactions with
a calcium atom present in a carbohydrate recognition domain (CRD). Many organisms
(plants, bacteria, virus and animals) use these proteins in various biological events
like lymphocyte adhesion, erythrocyte agglutination and extracellular matrix organization.
The C-type lectin fold is plastic and possible for about 1013 different sequences,
what promoted its adaptation to diverse functions, similarly to the observed for the
immunoglobulin fold (1014-1016 sequences). It is comprised of about 110-130 amino
acid residues that folds in two four-stranded β sheets sandwiched by two alpha helices.
Interestingly, C-type lectins present in snake venoms are possible anti-cancer agents
since they are toxic to cancer cells and inhibit the adhesion and proliferation of
various cancer cell lines. Therefore, we have purified a lactose binding C-type lectin
from the venom of Bothrops jararacussu (BJcuL) to study its structure and binding
properties to different sugars. BJcuL crystals were obtained by vapor diffusion and
the structure solved by X-ray crystallography to 2.9 Å resolution. BJcuL structure
is a decamer formed by a pseudo fivefold axis rotation of a dimer hold by a disulfide
bond. Each monomer binds a calcium atom and possibly another metal at a second and
opposed binding site. The decamer possesses a donut shaped structure with 10 calcium
ions on the surface available for interactions with carbohydrate molecules. Binding
specificity was evaluated for 20 carbohydrates using differential scanning fluorimetry
(DSF) that showed BJcuL interacts with galactose and lactose but less with glucose
and sacarose. Surprisingly, high levels of thermostabilization of BJcuL was achieved
with the antibiotic aminoglycosides geneticin (G418) and gentamicin in a calcium concentration
dependent manner, but not kanamycin. Intriguingly, while lactose and galactose inhibited
erythrocyte agglutination by BJcuL, G418 and gentamicin did not affect hemagglutination
implying a second site of binding. DSF analysis also suggested the presence of a second
binding site for the antibiotics and crystallization of the complexes are in progress
in order to understand fully this new binding mechanism of C-type lectin with antibiotics.
PL-050
Ab initio modelling of structurally uncharacterised antimicrobial peptides
Mara Kozic1
1Institute of Integrative Biology, University of Liverpool
Ab initio modelling of structurally uncharacterised antimicrobial peptides Mara Kozic
1* 1 Institute of Integrative Biology, Biosciences Building, University of Liverpool,
Crown Street, Liverpool L69 7ZB, United Kingdom * Mara.Kozic@liverpool.ac.uk Antimicrobial
resistance within a wide range of infectious agents is a severe and growing public
health threat. Antimicrobial peptides (AMPs) are among the leading alternatives to
current antibiotics, exhibiting broad spectrum activity. An understanding of the structure
of a protein can lead us to a much improved picture of its molecular function. Furthermore,
an improved understanding of structure-function relationships facilitates protein
design efforts to enhance their activity. Currently, the 3D structures of many known
AMPs are unknown. To improve our understanding of the AMP structural universe we have
carried out large scale ab initio 3D modelling of structurally uncharacterised AMPs.
Such ab initio modelling is facilitated by the typical small size of AMPs as well
as their tendency to contain disulphide bonds, these providing valuable additional
information to simulations. Preliminary results reveal unexpected similarities between
the predicted folds of the modelled sequences and structures of well-characterised
AMPs. For example, Lacticin Q was revealed to contain a helical bundle fold that bears
a striking resemblance to Enterocin 7A. We also found a remarkable similarity between
the predicted structure of Silkworm 001 peptide and β-hairpin AMPs such as Tachyplesin
I. Our results improve the understanding of the structure-function relationship of
AMPs.
PL-051
Surface aggregation-propensity as a constraint on globular proteins evolution
Susanna Navarro1, Marta Diaz2, Pablo Gallego2, David Reverter2, Salvador Ventura1
1Institut de Biotecnologia i Biomedicina and Departament de Bioquimica i Biologia,
2Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona
In living cells, functional protein–protein interactions compete with a much larger
number of nonfunctional interactions. Theoretical studies suggest that the three-dimensional
structures of present proteins have evolved under selective pressure to avoid the
presence of aggregation-prone patches at the surface that may drive the establishment
of anomalous protein contacts. However, no experimental evidence for this hypothesis
exists so far. The α-spectrin SH3 domain (SPC-SH3) has been used as a protein model
to decipher the sequential aggregation determinants of proteins. Here we use it to
address the structural determinants of protein aggregation and their link to protein
evolution. To this aim we exploit Aggrescan3D (A3D), a novel algorithm developed by
our group, which takes into account both protein structure and experimental data to
project aggregation propensities on protein surfaces. We used A3D to design a series
of SPC-SH3 variants with progressively stronger aggregation-prone surfaces and characterized
their thermodynamic, structural and functional properties. Our data support evolution
acting to constraint the aggregation propensities of globular protein surfaces in
order to decrease their potential cytotoxicity and the protein quality control machinery
acting to buffer this negative selective pressure.
PL-052
Utilizing 3D structure for the annotation of structural motifs in the Conserved Domain
Database
Narmada Thanki-Cunningham1, Noreen Gonzales1, Gabriele Marchler1, Myra Derbyshire1,
James Song1, Roxanne Yamashita1, Christina Zheng1, Stephen Bryant1, Aron Marchler-Bauer1,
Farideh Chitsaz11
1Conserved Domain Database, Structure Group CBB/NCBI/NLM/NIH
The Conserved Domain Database (CDD) is a protein classification and annotation resource
comprised of multiple sequence alignments representing ancient conserved domains.
CDD protein domain models are curated by NCBI and use 3D protein structure explicitly
to define domain extent and the location of conserved core structures, and to provide
accurate alignments between diverse family members via structure superposition. CDD
also imports external collections such as Pfam and TIGRFAM. Recently, a novel class
of annotation labeled as “structural motifs” has been introduced to supplement current
capabilities. These annotations define compositionally-biased and/or short repetitive
regions in proteins, which are difficult to model as functional domains conserved
in molecular evolution. Structural motifs include transmembrane regions, coiled coils,
and short repeats with variable copy numbers. For many types of short tandem repeats,
a few position-specific score matrices (PSSMs) suffice to annotate more than 90% of
the known instances of that structural motif. Unfortunately, a lack of sequence similarity
within coiled-coil regions prohibits the development of only a few generic models;
therefore, models for coiled-coil regions in the context of specific families have
been developed using the Spiricoil Database as a reference. Increased coverage of
coiled-coil regions in CDD, specific site annotations of these structural motifs as
well as their representation on the webpages will be discussed.
PL-053
Biophysical characterization of the Sema3A C-terminal basic domain interaction with
glycosaminoglycans
Roman Bonet1, Miriam Corredor1, Cecilia Domingo1, Jordi Bujons1, Yolanda Perez1, Ignacio
Alfonso1, Angel Messeguer1
1Department of Chemical Biology and Molecular Modelling, IQAC-CSIC
Semaphorin 3A (Sema 3A) is a protein originally described as an axonal chemorepellent
cue involved in many physiological processes ranging from embryonic development to
bone homeostasis or immune responses [1]. Sema3A signal transduction requires the
formation of a heteromeric complex with Neuropilin-1 (Nrp1) and PlexinA [2]. In addition,
Sema3A interaction with Nrp1 is modulated by the furin protease cleavage at its C-terminal
basic domain [3]. This C-terminal basic domain has also been suggested to mediate
the binding to glycosaminoglycans (GAGs), an association that locates Sema3A to perineuronal
nets and enhances its function in restricting neuronal plasticity and inhibiting axonal
regeneration in the central nervous system (CNS) [4, 5]. In this work, we used a combination
of biophysical techniques to gain insight into the interaction of the Sema3A C-terminal
domain with GAGs. Two peptides corresponding to the highly positively charged regions
on the domain were shown to bind to immobilized heparin by surface plasmon resonance
(SPR) and the affinity dramatically increased when the complete domain was assayed.
The binding was confirmed by nuclear magnetic resonance (NMR) and Circular Dichroism
(CD), which also revealed that the Sema3A C-terminus is mainly unstructured in solution
with a short helix in its N-terminus, as previously described for Sema3F [6]. The
conserved cysteine within this motif, necessary for the dimerization of Sema3A [7],
is also critical for the helix formation. In addition, fluorescence spectroscopy studies
showed that the N-terminal region also has a contribution in the binding to GAGs.
We acknowledge the financial support from the European Union Seventh Framework Programme
(FP7/2007-2013) under the Project VISION, Grant Agreement n° 304884.
1. Xu, R., Semaphorin 3A: A new player in bone remodeling. Cell Adh Migr, 2014. 8(1):
p. 5-10.
2. Janssen, B.J., et al., Neuropilins lock secreted semaphorins onto plexins in a
ternary signaling complex. Nat Struct Mol Biol, 2012. 19(12): p. 1293-9.
3.Parker, M.W., et al., Furin processing of semaphorin 3F determines its anti-angiogenic
activity by regulating direct binding and competition for neuropilin. Biochemistry,
2010. 49(19): p. 4068-75.
4. De Wit, J., et al., Semaphorin 3A displays a punctate distribution on the surface
of neuronal cells and interacts with proteoglycans in the extracellular matrix. Mol
Cell Neurosci, 2005. 29(1): p. 40-55.
5.Dick, G., et al., Semaphorin 3A binds to the perineuronal nets via chondroitin sulfate
type E motifs in rodent brains. J Biol Chem, 2013. 288(38): p. 27384-95.
6.Guo, H.F., et al., Mechanistic basis for the potent anti-angiogenic activity of
semaphorin 3F. Biochemistry, 2013. 52(43): p. 7551-8.
7.Koppel, A.M. and J.A. Raper, Collapsin-1 covalently dimerizes, and dimerization
is necessary for collapsing activity. J Biol Chem, 1998. 273(25): p. 15708-13.
PL-054
Functional clustering of the crotonase superfamily
Julia Hayden1, Janelle Leuthaeuser2, Patricia Babbit3, Jacquelyn Fetrow4
1Dickinson College, Molecular Biology and Chemistry Department, 2Department of Molecular
Genetics, Wake Forest University, 3Department of Pharm. Chem., University of California,
San Francisco, CA, 4Department of Chemistry, University of Richmond, Richmond, VA
As the number of sequenced proteins have grown, the reliance on computational annotation
has likewise grown, leading to rampant misannotation and difficulties grouping proteins
functionally. Prior attempts to create functionally relevant groupings of proteins
in the Crotonase superfamily suggest that this superfamily is difficult to cluster
functionally due in part to the functionally diverse nature of the protein superfamily.
We have developed two novel procedures to combat this difficulty: TuLIP (Two-Level
Iterative clustering Process), a process that utilizes structural information from
active sites to cluster protein structures into hypothesized functional groupings,
and MISST (Multi-level Iterative Sequence Searching Technique), a process that uses
the protein groupings created in TuLIP as a starting point for iterative GenBank searches
and further clustering after each search. Through these two methods, the total coverage
of the Crotonase superfamily has increased, and the generated groups contain proteins
from subgroups and families that did not have a structural representative. Novel hypothesized
functional protein groupings have been created, most notably for a large number of
proteins that lack annotation data at the subgroup or family level, and for proteins
of the enoyl-CoA hydratase family. Our results demonstrate the novel processes TuLIP
and MISST are able to cluster proteins of the Crotonase superfamily into hypothesized
functional groupings.
PL-055
Peptidic probes for intravascular molecular imaging of inflammation using clinically
translatable polymeric microbubbles
Olga Iranzo1,2, Ana C. Fernandes1, Teresa Sorbo2, Ivan Duka2, Lia Christina Appold3,
Marianne Ilbert4, Fabian Kiessling3, Ricardo J. F. Branco5
1Instituto de Tecnologia Química e Biológica António Xavier, UNL, 2Aix Marseille Université,
Centrale Marseille, CNRS, iSm2 UMR 7313, 3ExMI, Helmholtz Institute for Biomedical
Engineering, RWTH-Aachen University, 4BIP, IMM, Aix Marseille Université, CNRS, UMR
7281, 5UCiBio-REQUIMTE, Faculdade de Ciências e Tecnologia, UNL
E-selectin is a cell-adhesion molecule induced on the surface of endothelial cells
in response to cytokines. Its upregulation has been reported in many disorders, including
inflammatory and cardiovascular diseases, tumor angiogenesis and metastasis [1]. This
profile suggests E-selectin as a promising target to develop molecular imaging probes
for the detection of these diseases. Recently, we have reported the specific in vivo
ultrasound imaging of E-selectin expression in tumors using a microbubble contrast
agent covalently attached to the peptide ligand IELLQAR, known to bind to E-selectin
[2]. However, it was observed that this probe has a limitation in the imaging of cardiovascular
diseases where higher shear stresses prevent microbubbles from remaining attached
to the target. Therefore, peptides with higher E-selectin affinity are needed to design
probes capable of imaging these diseases. In this context, automated docking and molecular
dynamics methodologies were combined and applied to different E-selectin binding peptides.
These studies predicted the energetically more favorable binding mode as well as the
key interactions between the peptide ligands and the E-selectin receptor. Some of
these peptides were prepared by solid-phase peptide synthesis and their interactions
with E-selectin analyzed by surface plasmon resonance technique. The results showed
that these peptides have different affinities for E-selectin. These data were correlated
with the computational studies and evaluated to obtain crucial information of the
key recognition elements needed for higher E-selectin affinity. These recent results
will be presented.
[1] E. Jubeli, L. Moine, J. Vergnaud-Gauduchon, and G. Barratt, J. Control. Release
2012, 158, 194-206.
[2] S. Fokong, A. Fragoso, A. Rix, A. Curaj, Z. Wu, W. Lederle, O. Iranzo, J. Gätjens,
F. Kiessling, and M. Palmowski, Invest. Radiol. 2013, 48, 843-850.
PL-056
A search for anti-melioidosis drug candidates targeted to sedoheptulose-7-phosphate
isomerase from Burkholderia pseudomallei
Jimin Park1, Daeun Lee1, Sang A Yeo1, Mi-Sun Kim1, Dong Hae Shin1
1College of Pharmacy, Global Top5 Research Program, Ewha Womans University
Burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive
disease of animals and humans in tropical and subtropical areas. Sedoheptulose-7-phosphate
isomerase from B. pseudomallei (BpGmhA) is the antibiotics adjuvant target for melioidosis.
In general, BpGmhA converts d-sedoheptulose-7-phosphate to d-glycero-α-d-manno-heptopyranose-7-phosphate
(M7P). This is the first step of the biosynthesis pathway of NDP-heptose responsible
for a pleiotropic phenotype. Therefore, this biosynthesis pathway is the target for
searching novel antibiotics increasing the membrane permeability of Gram-negative
pathogens or adjuvants synergistically working with known antibiotics. The crystal
of this enzyme has been solved at 1.9 Å resolution. There is an active site pocket
where a putative metal binding site is located. To find out inhibitors of BpGmhA,
in-silico virtual screening with ZINC, a free database of commercially-available compounds,
has been performed. Tens of thousands of chemical compounds were docked into the active
site of BpGmhA. A number of putative BpGmhA binding compounds better than M7P were
found using Surflex-Dock included in the SYBYL software package. Characteristics of
these compounds were surveyed and classified to identify common binding properties
with BpGmhA.
PL-057
Mapping the structure of laminin using cross-linking and mass spectrometry
Gad Armony1, Toot Moran1, Yishai Levin2, Deborah Fass1
1Weizmann Institute of Science, Department of Structural Biology, 2Weizmann Institute
of Science, Israel Center for Personalized Medicine
Laminin, a ∼800 kDa heterotrimer, is a major element in the extracellular matrix (ECM).
Within the ECM, laminin contributes to the adhesion and migration of cells, both in
health and disease. The laminin trimer was observed by rotary shadowing electron microscopy
to be cross shaped: the three short arms of the cross are formed by the amino-terminal
halves of the three subunits, whereas the long arm of the cross holds the three chains
together in a long coiled coil. The narrow and flexible arms of the laminin cross
complicate studying its structure to high resolution by crystallography or electron
microscopy single particle reconstruction. To advance our understanding of this remarkable
quaternary structural assembly, we have used cross-linking and mass spectrometry to
analyze the organization of the laminin trimer. This technique was validated by known
crystal structures of isolated laminin domains. In all cases the crystal structure
distances agree with the cross-linker length. The identified cross-links were particularly
helpful in assigning the register and the subunit order of the long coiled coil due
to the high content of cross-linkable residues in this region. Using known X-ray crystal
structures, homology modeling, and distance restraints provided by two cross-linker
chemistries, a clearer picture of the laminin quaternary structure is obtained.
PL-058
Non-sequential protein structure alignment program MICAN and its applications
Shintaro Minami1, George Chikenji2, Motonori Ota1
1Dept. of Info. Sci., Nagoya Univ., 2Dept. of Comp. Schi. & Eng., Nagoya Univ.
In some proteins, secondary structure elements are arranged spatially in the same
manner, but they are connected in the alternative ways. Analysis on such non-sequential
structural similarity in proteins is important because it provides a deeper understanding
of the structural geometry of protein. This can be also observed even in the homologous
proteins, indicating the non-sequential structural similarity is significant in the
protein evolution. However, the non-sequential structural similarity in proteins is
less investigated. We developed a novel non-sequential structural alignment program
MICAN, which can handle Multiple chains, Inverse direction of chains, C$\alpha$models,
Alternative alignments, and Non-sequential alignments. We performed comprehensive
non-sequential structural comparison among homologous proteins in the same SCOP superfamily
by using the MICAN program. Based on the result, we found that approximately 8% of
superfamilies include at least one protein pairs showing non-sequential structural
similarity. 85% non-sequential structurally similar pairs are aligned in a simple
way, e.g. circular permutation, $\β$strand flip/swap, but 15% are complicated. Interestingly,
most of such complicated non-sequential similarities can be explicable by combination
of 2-4 simple non-sequential relationships. This result indicates that accumulation
of simple structural changes in the course of protein evolution produces completely
different fold homologs.
PL-059
Effects of cell-like infrastructures on transient protein interactions
Ciara Kyne1, Peter Crowley1
1School of Chemistry, National University of Ireland Galway
As early as 1919, Ritter surmised that the cell’s molecules cooperate to form a “special
apparatus and an organised laboratory”. 1 Despite supporting evidence from Srere,
McConkey and others, efforts to understand molecular organisation in vivo are still
in their infancy. However, important aspects of the cell interior have already been
revealed. For example, weak molecular interactions structure the cytoplasm into time-evolving,
functional zones. 2 Weak interactions are difficult to capture and can preclude protein
detection in cells by many biophysical techniques, including NMR spectroscopy. 3,
4 We explored the effects of cell-like milieus on the cytochrome c (cyt c)-flavodoxin
(fld) interaction. These oppositely charged proteins interact weakly with a number
of cognate partners. Neither cyt c4 nor fld is detectable by NMR in Escherichia coli
confirming their “sticky” nature (Figure 1A). The cyt c-fld interaction was assessed
in buffer, 8% polyacrylamide gels and in solutions containing 100 g/L of macromolecular
crowders (Figure 1B). 1H, 15N HSQC NMR revealed that the interaction was transient
in buffer, proceeding via the known binding site for both proteins. Substantial line
broadening was effected in crowded and confined solutions suggesting that the cyt
c-fld complex is stabilised under native-like conditions. The stabilising effect of
macromolecular crowders was also observed by native gel electrophoresis and crystallization.
These findings coincide with Spitzer and Poolman’s model for cytoplasmic structuring,
emphasising the role of charge-charge interactions and crowding in the formation of
macromolecular “clusters”. 5 The implications for cytoplasmic structuring will be
discussed alongside related investigations of cationic protein interactions in E.
coli extracts. 3, 4
Figure 1.
1H, 15N HSQC spectra of: A. E. coli cell suspensions containing over-expressed cyt
c and fld. B. cyt c with 1 equivalent of fld in media mimicking the cytoplasm. These
include 8% polyacrylamide gel, 100 g/L bovine serum albumin or polyvinylpyrrolidone
40, and buffer alone for comparison. Electrostatic surface representations of the
proteins are shown with their in-cell spectrum.
1.W. E. Ritter, R. G. Badger, Boston, 1919.
2.O. Medalia et al., Science 2002, 298, 1209.
3.C. Kyne et al., Prot. Sci. 2015, 24, 310.
4.P. B. Crowley et al., ChemBioChem 2011, 12, 1043.
5. J. J. Spitzer et al., Trends Biochem. Sci. 2005, 30, 538.
PL-060
A search for anti-melioidosis drug candidates targeted to D-glycero-D-manno-heptose-1,7-bisphosphate
phosphatase from Burkholderia pseudomallei
Jimin Park1, Sang A Yeo1, Daeun Lee1, Mi-Sun Kim1, Dong Hae Shin1
1College of Pharmacy, Global Top5 Research Program, Ewha Womans University
Burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive
disease of animals and humans in tropical and subtropical areas. D-glycero-D-manno-heptose-1,7-bisphosphate
phosphatase from B. pseudomallei (BpGmhB) is the antibiotics adjuvant target for melioidosis.
In general, BpGmhB converts D-glycero-D-manno-heptose-1β,7-bisphosphate to D-glycero-D-manno-heptose-1β-phosphate.
This is the third step of the biosynthesis pathway of NDP-heptose responsible for
a pleiotropic phenotype. Therefore, this biosynthesis pathway is the target for inhibitors
increasing the membrane permeability of Gram-negative pathogens or adjuvants synergistically
working with known antibiotics. To find inhibitors of BpGmhB, we performed homology
modeling of BpGmhB and in-silico virtual screening with ZINC, a free database of commercially-available
compounds. Tens of thousands of chemical compounds were docked into the active site
of BpGmhB. A number of putative BpGmhB binding compounds better than D-glycero-D-manno-heptose-1β,7-bisphosphate
were found using Surflex-Dock included in the SYBYL software package. Characteristics
of these compounds were surveyed and classified to identify common binding properties
with BpGmhB.
PL-061
Crystal structure of dimeric D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase
from Burkholderia thailandensis
Jimin Park1, Mi-Sun Kim1, Daeun Lee1, Keehyung Joo2, Gil-Ja Jhon3, Jooyoung Lee2,
Dong Hae Shin1
1Collegy of Pharmacy, Global Top5 Research Program, Ewha Womans University, 2School
of Computational Sciences, Korea Institute for Advanced Study, 3Department of Chemistry
and Nano Science, Ewha Womans University
We have solved the crystal structures of D-glycero-D-manno-heptose-1,7-bisphosphate
phosphatase from Burkholderia thailandensis (BtGmhB) catalyzing the removal of the
phosphate at the 7 position of D-glycero-D-manno-heptose-1,7-bisphosphate. It belongs
to the haloacid dehalogenase (HAD) superfamily with an α/β Rossman fold composed of
six parallel β-strands sandwiched between two sets of three α-helices. In solution,
BtGmhB presents both as monomer and dimer. The crystal structure of BtGmhB revealed
a dimeric form without catalytically important metal ions. This metal-free BtGmhB
shows the importance of zinc and magnesium ions for both structural and functional
reasons. It also contains one disulfide bond mediated by Cys95. A biochemical study
shows that this disulfide bond may be not essential for dimerization but responsible
for enzyme inactivation due to the absence of catalytic metal ions. A further biochemical
study is going on to identify a biorelevance of active and inactive dimeric forms
coupled to oxidation.
PL-062
Refined crystal structure of predicted fructose-specific enzyme IIB(fruc) from E.
coli
Jimin Park1, Daeun Lee1, Mi-Sun Kim1, Keehyung Joo2, Gil-Ja Jhon3, Jooyoung Lee2,
Dong Hae Shin1
1Collegy of Pharmacy, Global Top5 Research Program, Ewha Womans University, 2School
of Computational Sciences, Korea Institute for Advanced Study, 3Department of Chemistry
and Nano Science, Ewha Womans University
We have solved the crystal structures of predicted fructose-specific enzyme IIB(fruc)
from Escherichia coli (EcEIIB(fruc)). EcEIIB(fruc) belongs to a sequence family with
more than 5,000 sequence homologues with 25∼99% amino-acid sequence identity. It reveals
a conventional Rossman-like α-β-α sandwich fold with a novel β-sheet topology. Its
C-terminus is longer than its closest relatives and forms an additional β-strand whereas
the shorter C-terminus is random coils in the relatives. Interestingly, its core structure
is similar to that of enzyme IIB(cellobiose) from E. coli (EcIIB(cel)) transferring
a phosphate moiety. In the active site of the closest EcEIIB(fruc) homologues, a unique
motif CXXGXAHT comprising a P-loop like architecture including a histidine residue
is found. The conserved cysteine on this loop may be thiolated to act as a nucleophile
similar to that of EcIIB(cel). The conserved histidine residue is presumed to accommodate
negatively charged phosphate during enzymatic catalysis. Therefore, we propose that
the catalytic mechanism of EcEIIB(fruc) is similar to that of EcIIB(cel) transferring
phosphoryl moiety to a specific carbohydrate.
PL-063
NMR studies of the insertase BamA in three different membrane mimetics
Leonor Morgado1, Kornelius Zeth1,2,3, Björn M. Burmann1, Timm Maier1, Sebastian Hiller1
1Biozentrum, University of Basel, 2Department of Biochemistry, University of the Basque
Country, 3Ikerbasque - Basque Foundation for Research
β-barrel membrane proteins are ubiquitous in the outer membrane of Gram-negative bacteria,
mitochondria and chloroplasts, and are essential for transport, protein biogenesis,
cellular adhesion, and other cellular processes. In bacteria, outer membrane proteins
are inserted into the outer membrane by the β-barrel assembly machinery (Bam) (1).
This complex is composed of five proteins: the central unit BamA and the lipoproteins
BamB, BamC, BamD and BamE. BamA is a β-barrel membrane protein with five periplasmic
N-terminal polypeptide transport associated (POTRA) domains. The BamA structure has
been determined recently by X-ray crystallography (2,3), however its functional mechanism
is not well understood. This mechanism comprises the insertion of substrates from
a dynamic, chaperone-bound state into the bacterial outer membrane, and NMR spectroscopy
is thus a method of choice for its elucidation. In this work, BamA was reconstituted
and characterized in three membrane mimetic systems: LDAO micelles, DMPC:DiC7PC bicelles
and MSP1D1:DMPC nanodiscs. The impact of biochemical parameters on the NMR spectral
quality was investigated, including the total protein concentration and the detergent:protein
ratio. The transmembrane β-barrel of BamA is folded in either micelles, bicelles or
nanodiscs, however an N-terminally attached single POTRA5 domain is flexibly unfolded,
due to the absence of stabilizing contacts with other protein domains. Measurements
of backbone dynamics show distinct time scales of dynamic behavior for BamA β-barrel
and parts of its extracellular loop L6, revealing high local flexibility within the
the lid loop. This work presents the first high-resolution 2D solution NMR spectra
of the BamA barrel and establishes improved biochemical preparation schemes, which
will serve as a platform for structural and functional studies of BamA and its role
within the Bam complex.
References:
1. Rigel, NW, Silhavy, TJ (2012) Curr Opin Microbiol, 15, 189.
2. Noinaj, N, et al. (2013) Nature, 501, 385.
3. Ni, D, et al. (2014) FASEB J, 28, 2677.
PL-064
Biochemical characterization of the substrate specificity of two unique members of
the mammalian protein arginine methyltransferase family, PRMT7 and PRMT9
Andrea Hadjikyriacou1, You Feng1, Yanzhong Yang2, Mark Bedford3, Steven Clarke1,4
1Department of Chemistry and Biochemistry, University of California Los Angeles, 2Department
of Radiation Biology, Beckman Research Institute, City of Hope, 3Department of Epigenetics
and Molecular Carcinogenesis UT MD Anderson Cancer, 4Molecular Biology Institute,
University of California Los Angeles
Protein arginine methylation is a widespread and important posttranslational modification
in eukaryotic cells, shown to be involved in the activation or repression of transcription,
modification of the splicing machinery, signaling, and DNA repair. Mammalian protein
arginine methyltransferases include a family of nine sequence-related enzymes that
transfer one or two methyl groups onto the terminal guanidino groups on arginine residues,
producing monomethylarginine only (MMA, type III), symmetric dimethylarginine (SDMA)
and MMA (Type II), or asymmetric dimethylarginine (ADMA) and MMA (Type I). While PRMT1,
2, 3, 4, 6, and 8 have been characterized as type I enzymes, and PRMT5 as a type II
enzyme, the role and activity types of the two final members of this family of enzymes,
PRMT7 and PRMT9, had been unclear due to conflicting results in the literature, and
the substrates for these enzymes had been elusive. Both PRMT7 and PRMT9 are distinct
members of the family with two methyltransferase or methyltransferase-like domains
and containing acidic residues in otherwise well-conserved substrate double E binding
motif, features not seen in the other PRMT enzymes. Recent work in our laboratory
confirmed PRMT7 as the only type III MMA-forming enzyme in the group, with a unusual
low temperature optimum for activity, and a heretofore not seen preference for a basic
stretch of residues in an R-X-R sequence for methylation. Mutations of the acidic
residues in the substrate-binding motif results in a loss of the specific R-X-R activity
and the appearance of a G-R-G specificity typical of many of the other PRMTs. The
physiological substrate of PRMT7 has yet to be confirmed, although histone H2B is
an effective in vitro substrate. PRMT9, on the other hand, had no reported activity,
until immunoprecipitation from HeLa cells showed it pulled down two splicing factors,
SF3B2 and SF3B4, in a complex. Amino acid analysis showed that PRMT9 methylates SF3B2
to produce both MMA and SDMA, thus making it the second type II enzyme in mammals.
PRMT9 knockdown results in modulation of alternative splicing events. This enzyme
appears to be relatively specific for the SF3B2 protein; a peptide containing the
methylatable arginine residue was not found to be a substrate, and typical substrates
of other PRMTs are not recognized by PRMT9. We found that the position of the methylated
arginine residue in SF3B2 is important, and the acidic residues in the substrate-binding
motif also play an important role in substrate recognition. Thus, PRMT7 and PRMT9
represent unique members of the mammalian PRMT family.
PL-065
Ornithine decarboxylase participates in autophagy by ultraviolet Binduced cell injury
Guang-Yaw Liu1, Yen-Hung Lin2, Hui-Chih Hung2
1Institute of Microbiology & Immunology, Chung Shan Medical University, and Divi,
2Department of Life Sciences, National Chung Hsing University (NCHU)
Ornithine decarboxylase (ODC) plays an essential role in various biological functions,
including cell proliferation, differentiation and cell death. In this study, we revealed
that overexpression of ODC in HeLa and MCF-7 cells decreased cellular ROS (Reactive
oxygen species) after low dose of ultraviolet B radiation (UVB), leading autophagy
inhibited, and it was restored by knocking down ODC (shODC) in ODC overexpressing
HeLa and MCF-7 cells. Furthermore, the results demonstrated that AMPK was increased
after high dose of UVB radiation in ODC ovexpressing HeLa and MCF-7 cells, leading
autophagy induced and apoptosis inhibited. We demonstrated that knocked down autophagy
by shRNA (shAtg5, shBECN1, and shAtg12) and chloroquine (CQ) could enhance high dose
of UVB induced cell death in ODC overexpressing HeLa and MCF-7 cells. Here, we also
observed that knocked down ODC in ODC overexpressing HeLa and MCF-7 cells inhibited
autophagy and enhanced high dose of UVB radiation. Because of Atg12 can regulate cell
apoptosis and utophagy. Site directed mutagenesis was used to mutant the amino acid
which can regulate cell apoptosis and autophagy on Atg12, respectively in these two
ODC overexpressing cells. According to the results, mutated the amino acid which can
regulate apoptosis on Atg12 leading the cells more survival. Relatively, mutated the
amino acid which can regulate autophagy on Atg12 leading the cells died. Therefore,
inhibition of ODC and autophagy could be a promising strategy for adjuvant chemotherapy
in human breast and cervical cancers.
Keywords:
Ornithine decarboxylase, Autophagy, Apoptosis, Ultraviolet B
PL-066
Fish ß-parvalbumin acquires allergenic properties by amyloid assembly
Javier Martínez1, Rosa Sánchez1, Milagros Castellanos2, Ana M. Fernández-Escamilla3,
Sonia Vázquez-Cortés4, Montserrat Fernández-Rivas4, María Gasset1
1Instituto Química-Física ’Rocasolano’, CSIC, 2Centro Nacional de Biotecnología, CSIC,
3Estación Experimental del Zaidín, CSIC, 4Allergy Department, Hospital Clínco San
Carlos, IdISSC
Principles: Amyloids are highly cross-β-sheet-rich aggregated states that confer protease
resistance, membrane activity and multivalence properties to proteins, all essential
features for the undesired preservation of food proteins transiting the gastrointestinal
tract and causing type I allergy. Methods: Amyloid propensity of β-parvalbumin, the
major fish allergen, was theoretically analyzed and assayed under gastrointestinal
relevant conditions using the binding of thioflavin T, the formation of SDS-resistant
aggregates, circular dichroism spectroscopy and atomic force microscopy fibril imaging.
Impact of amyloid aggregates on allergenicity was assessed by dot blot. Results: Sequences
of β-parvalbumin of species with commercial value contain several adhesive hexapeptides
capable of driving amyloid formation. Using Atlantic cod β-parvalbumin (rGad m 1)
displaying high IgE crossreactivity, we have found that formation of amyloid fibers
under simulated gastrointestinal conditions accounts for the resistance to acid and
neutral proteases, for the presence of membrane active species at gastrointestinal
relevant conditions and for the IgE-recognition in allergic patient sera. Incorporation
of the anti-amyloid compound epigallocathequin gallate prevents rGad m1 fibrillation,
facilitates its protease digestion and impairs its recognition by IgE. Conclusions:
rGad m 1 amyloid formation explains its degradation resistance, its facilitated passage
across the intestinal epithelial barrier and the epitope architecture as allergen.
Financial Support:
This work was partially supported by Ministerio de Economía y Competitividad (BFU2009-07971
and SAF2014-52661 to MG, BIO2011-28092 and CSD2009-00088 to MC), Fundación CIEN (MG)
and Raman Health (MG)
PL-067
Peptidylarginine Deiminase 2 Assigns Activated T Cell Autonomous Death
Guang-Yaw Liu1, Wen-Hao Lin2, Hui-Chih Hung2
1Institute of Microbiology & Immunology, Chung Shan Medical University, and Divis,
2Department of Life Sciences, National Chung Hsing University (NCHU)
Peptidylarginine deiminase type 2 (PADI2) is a protein post-translational modification
enzyme that catalyzes arginine residues into the citrulline residues. Previous studies
have been shown that PADI2 promotes protein citrullinations in lymphocytes and it
could plays an important role in inflammation. We found that overexpression of PADI2
promotes apoptosis in activated T cells previously. Whether PADI2 participate in the
pathway of activated T cell autonomous death (ACAD) is still curious. In the delicate
PADI2-mediated ACAD, we found that overexpression of PADI2 displayed the higher levels
of citrullinated protein which induced the ER stress significantly. The high levels
of citrullinated protein results unfolding protein response (UPR) of ER stress and
increases the loading of protein degradation. Autophagy could degrade the citrullinated
and unfolding protein. Herein, PADI2 could enhance autophagy in Jurkat T cells and
lead to a degradation of p62 and the accumulation of LC3-II. Autophagy and apoptosis
are two critical mechanisms which participate against cellular stress, cell activation,
survival and homeostasis. PAD2-overexpressed Jurkat T cells caused the activation
of Th17 cells to increase mRNA expression of cytokines, such as IL-17, IL-21, IL-22
and TNFα. Cytokines provoked caspase expression and led to caspase-mediated cleavage
of Beclin-1 which was an important factor of apoptotic signaling. Knockdown of BCEN1
rescued cell survival due to the increase of Bcl-xL and the decrease of caspase-3.
We suggested that PADI2 participated in the activated T cell-induced autonomous death
through triggering ER stress pathway, stimulating the expression of cytokines and
promoting autophagy by PADI2-citrullinated protein.
Key words:
PADI2, ER stress, Autophagy, Cytokine, Activated T cell autonomous death (ACAD).
PL-068
Studies on secondary metabolites production and proteins and enzymes of in vitro cultivated
Artemisia alba Turra and relations with some endogenous phytohormones
Yuliana Raynova1, Krassimira Idakieva1, Vaclav Motyka2, Petre Dobrev2, Yuliana Markovska3,
Milka Todorova1, Antoaneta Trendafilova1, Ljuba Evstatieva4, Evelyn Wolfram5, Kaliva
Danova1
1Institute of Organic Chemistry with Centre of Phytochemistry, BAS, 1113 Sofia, B,
2Institute of Experimental Botany, CAS, Prague, Czech Republic, 3Faculty of Biology,
Sofia University “St. Kliment Ohridski”, Sofia 1164, Bulgari, 4Institute of Biodiversity
and Ecosystem Research, BAS, 1113 Sofia, Bulgaria, 5Zurich University of Applied Sciences,
Institute of Biotechnology, Phytopharmacy
Studies on secondary metabolites production and proteins and enzymes of in vitro cultivated
Artemisia alba Turra and relations with some endogenous phytohormones Yuliana Raynova1,
Krassimira Idakieva1, Vaclav Motyka2, Petre Dobrev2, Yuliana Markovska3, Milka Todorova1,
Antoaneta Trendafilova1, Ljuba Evstatieva4, Evelyn Wolfram5, Kalina Danova1 1 Institute
of Organic Chemistry with Centre of Phytochemistry, BAS, 1113 Sofia, Bulgaria 2 Institute
of Experimental Botany, CAS, Prague, Czech Republic 3 Faculty of Biology, Sofia University
“St. Kliment Ohridski”, Sofia 1164, Bulgaria 4 Institute of Biodiversity and Ecosystem
Research, BAS, 1113 Sofia, Bulgaria 5 Zurich University of Applied Sciences, Institute
of Biotechnology, Phytopharmacy, Wädenswil, Switzerland Aim: Artemisia alba Turra
is an essential oil bearing shrub, characterized with great variability of the essential
oil profile of wild grown plants, related to genetic, geographic and environmental
factors. It was previously established that inhibition of rooting in vitro caused
by cytokinin/auxin treatment affected the essential oil profile of the plant and these
changes were also related to bioactive endogenous cytokinin levels in vitro (1, 2).
The aim of the present work was to perform a complex study on the relations between
soluble protein levels, enzyme activities (studied spectrophotometrically and by SDS-PAGE
and zymography); malondialdehyde and hydrogen peroxide levels, endogenous hormones
(cytokinins, salycilic acid, as well as jasmonic acid and its conjugates), polyphenolics
and terpenoids in a model system of A. alba in vitro with inhibition of rootng and
stimulation of callusogenesis by means of individual and combined cytokinin and cytokinin/auxin
treatments. Results: It was established that inhibition of rooting and stimulation
of callusogenesis caused by benzyl adenine (BA) or combinations of BA and indole-3-butiric
acid (IBA) in vitro were related to elevation of sesquiterpenoids in the essential
oils, as well as polyphenolics content, accompanied by a drop of stress hormones,
bioactive cytokinins and preservation of oxidative stress and lipid peroxidation levels,
as compared with non-treated control. Individual treatments with either IBA or BA,
also increased the sesquiterpenoid content in the essential oil of the plant, in a
concentration related manner, this effect being more profound after BA treatment.
In addition, BA treated plants exhibited a drop of protein levels of the aerial samples,
as well as profound differences of enzymatic activity in the callus tissues, as compared
with callus of plants treated with different combinations of BA and IBA. Conclusion:
The results of the present work indicate that alterations of endogenous phytohormonal
levels, caused by exogenous plant growth regulators treatment, might be the mediator
between primary and secondary metabolism by means of affecting protein levels and
activity of key enzymes in vitro.
Acknowledgements:
PhytoBalk, SNF No. IZEBZ0_142989 and SD-MEYS No. DO2-’01:153; MEYS CR, project No.
LD14120; bilateral cooperation project between BAS and CAS, Reg. No. 29.
References:
1. Danova K, Todorova M, Trendafilova A, Evstatieva L (2012) Cytokinin and auxin effect
on the terpenoid profile of the essential oil and morphological characteristics of
shoot cultures of Artemisia alba. Natural Product Communications 7: 1-2.
2. Krumova S, Motyka V, Dobrev P, Todorova M, Trendafilova A, Evstatieva L, Danova
K (2013) Terpenoid profile of Artemisia alba is related to endogenous cytokinins in
vitro. Bulgarian Journal of Agricultural Science, 19: 26–30.
PL-069
Evaluation of human salivary α-defensins by LC-ESI-MS
Nadia Ashrafi1, Cris Lapthorn1, Fernando Naclerio2, Frank Pullen1, Birthe Nielsen1,
Yue Fu2, Jack Miller2, Christian Watkinson2, Marcos Seijo2
1University of Greenwich (Faculty of Engineering and Science), 2University of Greenwich
(Centre for Sport Science and Human Performance)
Human neutrophil α-defensins (HNP 1-4) are small cationic, structurally homologous
peptides which play a central role not only in infection and inflammation but also
have direct antimicrobial activity against various bacteria, viruses, fungi, and parasites.
Human neutrophils are granulocytes that are predominantly found in the blood and may
account for as much as 70% of the total circulating leukocyte population. Several
study has demonstrated an increased concentration of HNPs in biological fluid (plasma,
blood, saliva, serum) of patients with lung diseases, crohn’s disease, uncreative
colitis, oral disease, gastric, type 1 diabetes and colorectal cancer. Salivary HNP
1-3 are conventionally measured using an enzyme-linked immunosorbent assay (ELISA)
which does not discriminate between individual HNPs due to their structural similarities.
Considering the biological importance of salivary human neutrophil α-defensin (HNPs),
there is therefore, a need to develop an analytical method that will discriminate
between the defensins. An LC-MS method has been established for the separation and
detection of HNP 1-4. The method has been optimised, validated and applied to examine
the relative level of HNP 1-4 in participants undertaking a circuit resistance training
workout. To date, no studies have systematically investigated the effect of acute
(min to hours) and chronic (days to weeks) change in salivary α-defensins family before
and after exercise by LC-ESI-MS. Twelve resistance trained athletes participated in
the study. Participants consumed a placebo or a multi-nutrient supplement during exercise
and the HNP 1-4 response was investigated pre, post 30 and 60 minutes of the workout.
The data reveals that the difference between HNP 1-4 levels pre and post exercise
were significant; p <0.03 for participants consuming either the placebo or the multi-nutrient
drinks. The increased HNP levels may be a part of the normal stress response. Supplementing
with the multi-nutrient drink resulted in decreased level of HNPs compared to placebo
data which indicates that the supplementing reduce exercise-induced airway inflammation.
The present work demonstrated that, HNP 1-4 could be used as potential stress biomarkers.
PL-070
Characterising interactions between alginates of different sizes and ß-lactoglobulin
Emil G. P. Stender1, Sanaullah Khan2, Outi E. Mäkinen3, Kristoffer Almdal2, Peter
Westh4, Richard Ibsen2, Maher Abou Hachem1, Birte Svensson1
1Technical University of Denmark – DTU – Department of Systems Biology, 2.−2Technical
University of Denmark – DTU – Department of Micro- and Nanotechnology, 3University
of Copenhagen – Department of Food Science, 4Roskilde University – RUC – Department
of Science, Systems and Models
Alginate is a polysaccharide from brown algae consisting of (1→4)-β-D-mannuronic acid
and α-L-guluronic acid (1). Here interaction is characterised between β-lactoglobulin
the most abundant whey protein and two alginates of different average molecular weight.
At pH below the pI of β-lactoglobulin large particles are formed whose hydrodynamic
diameter depends on pH as deduced from dynamic light scattering data (2). The binding
strength and stoichiometry as assessed at pH 3 and pH 4 by aid of isothermal titration
calorimetry showed no difference in dissociation constants at these pH values, while
the binding stoichiometry is increased 2.5 fold. Furthermore, the binding stoichiometry
varied 7 fold among the two alginates corresponding to their difference in average
molecular weight and in addition 20 fold higher binding affinity was found with the
high as compared to the low molecular weight alginate. In conclusion, the binding
stoichiometry of β-lactoglobulin with alginate increases by a factor that correlates
to the average molecular weight of the alginate and also a much higher affinity was
found for the high molecular weight alginate.
Acknowledgements:
This work is supported by the Danish Council for Strategic Research and by a PhD fellowship
from the Technical University of Denmark
1. Haug, A., Larsen, B., and Smidrod, O. (1966) Acta Chemica Scandinavia 20, 183-&.
2. Hosseini, S. M. H., Emam-Djomeh, Z., Razavi, S. H., Moosavi-Movahedi, A. A., Saboury,
A. A., Atri, M. S., and Van der Meeren, P. (2013) Food Hydrocolloids 32, 235-244.
PL-071
Validation of a LC-MS method for the detection of human salivary α-defensins
Nadia Ashrafi1, Cris Lapthorn1, Birthe Nielsen1, Fernando Naclerio2, Frank Pullen1,
Patricia Wright1
1University of Greenwich (Faculty of Engineering and Science), 2University of Greenwich
(Centre for Sport Science and Human Performance)
A LC-MS method for the detection of human salivary alpha-defensins (HNP1-4) in stimulated
whole saliva has been developed and validated which extend to sample preparation.
HNP 1-3 have almost identical amino acid sequences. HNP 1-4 differs in their N-terminal
amino acid residues. The presence of mucins and other high molecular weight glycoproteins
in saliva makes the direct analysis of defensins difficult. The LC-MS method was linear
for concentrations of HNP-2 between 0.05 and 1 ng/mL (R2 = 0.99) with a LOD of 0.05
ng/mL. Inter and intra assay precision was 0.94 – 15%, respectively. Saliva sample
were clean up by solid phase extraction (SPE) and without-solid phase extraction (WSPE)
method. Mobile phase composition was delivered at a flow rate of 0.6 mL/min for 3
mm internal diameter column and 0.3 mL/min for 2.10 mm internal diameter column in
relation to the method transfer. During LC-MS optimisation, two different mobile phases
compositions (MeOH: H2O; MeCN: H2O) with three different additives ((0.1% (v/v); formic
acid, acetic acid, ammonium format with formic acid) have been investigated in response
to ion intensity of ESI-MS for individual HNP 1-4 in saliva. Kinetex® column separation
efficiency was evaluated using two different column dimensions (50 x 2.1 mm and 50
x 3 mm.) and two different stationary phases (C18 and C8). Kinetex® column (homogenous
porous shell) performance was also compared to new ultra ACE® (encapsulated bonded
phase) column. Sample optimisation revealed that the SPE method removes interference
from salivary glycoproteins and consequently yields larger peak area (30-90%) for
all HNPs. HNPs were extracted by SPE with a recovery of 80-91%. The MeOH: H2O: acetic
acid (0.1%) provided enhanced (P>0.05) HNP1-3 ion intensities. The Kinetex® C8 (50
x 3.0 mm, 2.6 µm) column facilitated a better separation efficiency of the four HNPs
as compared to the Ultra Core Super C18 ACE® (50 x 3.0 mm, 25 µm) column, the Kinetex®
C18 (50 x 3.0 mm, 2.6 µm) and the Kinetex® C18 (50 x 3.0 mm, 5 µm) column. The relative
levels of the HNPs were determined in healthy volunteers before and after a rigorous
exercise regime: It is possible that prolonged strenuous exercise will affect oral
innate immunity and therefore also the level of salivary defensins. HNP1-3 are traditionally
detected in an enzyme-linked immunosorbent assay (ELISA) which does not discriminate
between the different HNPs due to their structural similarities. There has therefore
been a need to develop a mass spectrometry method that will discriminate between the
defensins. As part of the method validation, the HNP1-3 level was determined by ELISA
and the data was compared with the LC-MS data. Here we present this cross-validation;
the data revealed no significance difference between the two methods (R2= 0.96) which
confirms that the developed LC-MS method is and equal sensitive method for the detection
of these potential antimicrobial markers. This method can easily be adopted for similar
molecular weight of peptides as HNPs and also for any other biological matrix.
PL-072
Moonlighting proteins: relevance for biotechnology and biomedicine
Luis Franco Serrano1, Sergio Hernández1, Alejandra Calvo2, Gabriela Ferragut2, Isaac
Amela1, Juan Cedano2, Enrique Querol1
1Institut de Biotecnologia i Biomedicina. Universitat Autònoma de Barcelona, 2Laboratorio
de Inmunología, Universidad de la República Regional Norte-Salto
Multitasking or moonlighting is the capability of some proteins to execute two or
more biochemical functions. The identification of moonlighting proteins could be useful
for researchers in the functional annotation of new genomes. Moreover, the interpretation
of knockout experiments, in which the result of a gene knocking does not produce the
expected results, might be enhanced. The action of a drug can also be facilitated
because it might have an off-target or side effect with somewhat hidden phenotypic
traits. It would be helpful that Bioinformatics could predict this multifunctionality.
In the present work, we analyse and describe several approaches that use protein sequences,
structures, interactomics and current bioinformatics algorithms and programs to try
to overcome this problem. Among these approaches there are: a) remote homology searches
using Psi-Blast, b) detection of functional motifs and domains, c) analysis of data
obtained of protein-protein interaction databases (PPIs), d) matches of the sequence
of the query protein to 3D databases (i.e., algorithms like PISITE), e) mutation correlation
analysis between amino acids using algorithms like MISTIC. Remote homology searches
using Psi-Blast combined with data obtained from interactomics databases (PPIs) have
the best performance. Structural information and mutation correlation analysis can
help us to map the functional sites. Mutation correlation analysis can only be used
in very specific situations because it requires the existence of a multialigned family
of protein sequences, but it can suggest how the evolutionary process of second function
acquisition took place. We have designed a database of moonlighting proteins, MultitaskProtDB
(http://wallace.uab.es/multitask/). From this database we determine the frequencies
of canonical and moonlighting coupled functions (being an enzyme and a transcription
factor the highest), the percentage of moonlighting proteins involved in human diseases
(65% of the human moonlighting proteins in the database) and the percentage of moonlighting
proteins acting as a pathogen virulence factor (20% of the moonlighting proteins in
the database).
PL-073
Correlation between potential human neutrophil antimicrobial peptides (HNP 1-3) and
stress hormones in human saliva
Nadia Ashrafi1, Frank Pullen1, Birthe Nielse1, Cris Lapthorn1, Fernando Naclario2
1University of Greenwich (Faculty of Engineering and Sciene), 2University of Greenwich
(Centre of Sports Science and Human Performance)
Numerous studies have investigated the effect of exercise on mucosal immunity but
the focus has mainly been on salivary immunoglobulins lysozymes and hormones (cortisol,
testosterone). This is not surprising given that IgA and IgG are the predominant immunoglobulins
in saliva and there is a relationship between mucosal immunity and upper respiratory
illness. It is well known that physical and mental stress provoke the release of cortisol
from hypothalamic pituitary adrenal axis, by which stress can modulate various immune
responses. In general, cortisol and growth hormones helps to induce the activation
of neutrophils. To date, this study represents the first study that investigated the
correlation between human neutrophil alpha defensins family against cortisol (stress
hormone) and testosterone (growth hormone) in human saliva before and after exercise
or training. Twelve resistance trained athletes volunteered to participate in the
study. Participants consumed supplements during exercise and the HNP 1-3, cortisol
and testosterone response was investigated pre, post 30 and 60 minutes of the workout.
The correlation between salivary antimicrobial peptide (HNP 1-3) and stress hormone
(cortisol and testosterone) has been investigated using ELISA. Cortisol showed no
significant (p = 0.818) difference for (pre to 30 min post) between CHO and PL (CHO:
483.07 ± 912.77 ng/mL; PL= 583.82 ± 1134.33 ng/mL) conditions but a strong trend (p = 0.074)
was observed for (pre to 60 min post) post (CHO: 1023.19 ± 1500.40 ng/mL; PL= 1480.33 ± 2214.80
ng/mL) condition. Testosterone showed no significant (p = 0.167; p = 0.156) difference
for (pre to 30 min post) between CHO and PL (CHO: 23.32 ± 44.11 ng/mL; PL= 10.40 ± 14.19
ng/mL) and for (pre to 60 min post) post (CHO: 26.42 ± 19.11 ng/mL; PL= 23.72 ± 17.91
ng/mL) condition. HNP 1-3 showed no significant (p = 0.348) difference for (pre to
30 min post) between CHO and PL (CHO: 72.26 ± 148.82; PL= 125.20 ± 70.00) conditions
but significant difference (p = 0.026) was observed for (pre to 60 min post) between
CHO and PL (CHO: 35.18 ± 182.69; PL= 228.74 ± 151.63) condition. The present findings
suggested that there is no correlation between salivary HNP 1-3 and cortisol for (PL:
R2 = 0.02 and CHO: R2 = 0.01); HNP 1-3 and testosterone (PL: R2 = 0.20 and CHO: R2 = 0.10).
A worth note from previous study which suggested that using murine skin model (an
increase in endogenous glucorticoids (cortisol) by physiological stress reduced mRNA
levels of antimicrobial peptide (cathelicidin). It is not clear that the correlation
between hormones and antimicrobial peptide has been affected by the time interval
of the exercise. Both cortisol and antimicrobial peptide demonstrated a transient
increase after exercise but it is surprising that they are not correlate to each other.
One of the hypothesis from the present finding could be cortisol responses slow and
it will be interesting to do further research with longer interval. The second hypothesis
demands a further investigation to determine the synergism between substances.
PL-074
GWIDD: Genome-Wide Docking Database
Madhurima Das, Varsha D. Badal, Petras J. Kundrotas, Ilya A. Vakser
1Center for Computational Biology, The University of Kansas
GWIDD: GENOME-WIDE DOCKING DATABASE Madhurima Das, Varsha D. Badal, Petras J. Kundrotas
and Ilya A. Vakser Center for Computational Biology, The University of Kansas, Lawrence,
Kansas, USA Structural characterization of protein-protein interactions (PPI) is essential
for understanding molecular mechanisms in living systems. Genome-Wide Docking Database
(GWIDD) provides the most extensive data repository of structures and models of PPI
on a genomic scale. Currently, we are expanding the GWIDD dataset to 800,365 PPI in
1,652 organisms, up from 128,818 PPI in 771 organisms in the previous release. The
PPI data were imported from INTACT and BIOGRID databases and were subjected to in-house
modeling pipeline. GWIDD current implementation contains 11,073 experimentally determined
complexes, and 12,426 sequence homology and 28,811 structure homology models of complexes.
The user-friendly interface offers flexible organism-specific search with advanced
functions for a refined search for one or both proteins. The new GWIDD version includes
also a new interactive visualization screen that allows to view search results in
different residue representations with the emphasis on the PPI interface. GWIDD is
available at http://gwidd.compbio.ku.edu.
PL-075
Refolding and activation of recombinant trypsin i from sardine fish (sardinops sagax
caerulea)
Manuel Carretas-Valdez1, Francisco Cinco-Moroyoqui1, Marina Ezquerra-Brauer1, Enrique
Marquez-Rios1, Rogerio Sotelo-Mundo2, Idania Quintero-Reyes3, Aldo Arvizu-Flores3,
1Universidad de Sonora, Departamento de Investigación en Alimentos, 2Centro de Investigación
en Alimentación y Desarrollo, A.C., 3Universidad de Sonora, Departamento de Ciencias
Químico Biológicas
Trypsin (EC 3.4.21.4) is the principal member of the serine protease family, and catalyzes
the hydrolysis of proteins and peptides specifically at the carboxyl group of lysine
and arginine residues. The trypsin I studied from Monterey sardine (Sardinops sagax
caerulea) is described as a cold-adapted enzyme. Previous to evaluate structure-function
relationships by site-directed mutagenesis, we first established the experimental
conditions to perform the recombinant expression, purification, refolding and activation
for wild-type trypsin I from Monterey sardine. Trypsin I was overexpressed in E. coli
BL21 as a fusion protein of trypsinogen and thioredoxin, which was obtained in an
insoluble form. Inclusion bodies were extracted and disolved in urea 4 M and DTT 30
mM. Refolding was achieved with buffer Tris-HCl, 55 mM, pH 8.8, NaCl 264 mM, KCl 11
mM, polyethylenglycol 0.055%, GSSG 1 mM and GSH 5 mM. The recovery of the refolded
recombinant trypsinogen I was 29 mg per liter of LB medium. Before activation, the
45 kDa trypsinogen-thiorredoxin did not show trypsin-like activity against BApNA.
Activation was achieved by the addition of 0.01 U/mL of native trypsin I purified
from sardine pyloric caeca (non-recombinant). The activated recombinant trypsin showed
up to four times more activity than the non-recombinant trypsin. The described protocol
allowed us to obtain sufficient amounts of protein for further biochemical and biophysical
characterization. Therefore, the recombinant trypsin I from Monterey sardine is feasible
as a model for structure-function studies for cold-adapted proteins.
PL-076
WapA and SMU_63c are Amyloidogenic Proteins of Streptococcus mutans
Richard Besingi1, L. Jeannine Brady1
1Department of Oral Biology, University of Florida
We showed previously that adhesin P1 (Ag I/II) of the acidogenic bacterium Streptococcus
mutans, a causative agent of human tooth decay, is capable of amyloid fibrillization.
Known inhibitors of amyloid fibril formation inhibit biofilm formation by amyloidogenic
microbes, including S. mutans, thus suggesting a potential mechanism for therapeutic
intervention. Amyloid is detectable in human dental plaque and is produced by both
clinical and laboratory strains of S. mutans, further supporting a functional role.
S. mutans lacking P1 demonstrates residual amyloid forming properties, however, a
mutant lacking sortase, the transpeptidase which covalently links P1 and several other
proteins to the peptidoglycan cell wall, is defective in cell-associated amyloid-like
properties. The objectives of this study were to identify additional amyloid forming
proteins of S. mutans and to evaluate the effects of buffering conditions and pH on
the ability of the identified proteins to form amyloids. A P1-deficient mutant strain
was grown to stationary-phase in defined minimal media, and secreted proteins from
spent culture supernatants were fractionated by ion exchange chromatography. Partially
purified protein fractions were tested for binding of the amyloidophilic dyes Congo
Red (CR) and Thioflavin T (ThT), and for characteristic birefringent properties following
staining with CR and visualization under crossed polarizing filters. Proteins from
fractions that tested positive for amyloid-like material were separated by SDS PAGE,
and identified by LC/MS. These included WapA, GbpA, GbpB, SMU_2147c and SMU_63c. Recombinant
proteins were expressed in Escherichia coli, and purified for confirmation and characterization
of individual amyloidogenic properties in vitro. Recombinant WapA and SMU_63c displayed
all the biophysical characteristics of amyloid, including visualization of fibrillar
aggregates when viewed by transmission electron microscopy. In contrast, GbpA and
SMU_2147c produced amorphous aggregates. WapA and SMU_63c form amyloid at different
pH, SMU_63c under acidic conditions and WapA under neutral to basic conditions. This
suggests that the prevailing environmental pH may represent different in vivo triggers
for amyloid fibrillization of different S. mutans proteins. Genes encoding P1, WapA,
and SMU_63c have been deleted from the S. mutans genome individually and in combination
with each other. This will facilitate subsequent assessment of their individual susceptibilities
to amyloid inhibitors, their contributions to amyloid formation under varying environmental
conditions, and their respective roles in biofilm formation.
PL-077
Characterization of the membrane-localized interaction network between the GTP-ase
Rheb and the FKBP12-like protein FKBP38 by NMR
Maristella De Cicco1, Sonja A. Dames1
1The Affiliation Is Technische Universität München
Rheb is a homolog of Ras GTPase. Like other small GTPases, the activity of Rheb is
dictated by its guanine nucleotide binding states: it is active in its guanosine 5′-triphosphate
(GTP) bound form and inactive in the guanosine diphosphate (GDP)–bound form. Rheb
proteins play critical roles in regulating growth and cell cycle, and this effect
is due to its role in regulating the insulin/TOR/S6K signaling pathway [1-3]. Rheb
binds directly to a region N-terminal of the kinase domain in mTOR and activates it
in a GTP-dependent manner. C-terminal farnesylation allows Rheb to associate with
the endomembranes. Conditions preventing Rheb endomembrane localization impair its
ability to interact with the components of the mTOR complex 1 (mTORC1) to activate
downstream targets [4]. The heterodimeric Rag GTPases localize mTORC1 to lysosomes
by their amino-acid-dependent interaction with the lysosomal Ragulator complex. Rheb
is also thought to reside on lysosomes to activate mTORC1 [5]. Based on coimmunoprecipitation
and an in vitro binding assays Rheb regulates mTOR through FKBP38, a member of the
FK506-binding protein (FKBP) family that is structurally related to FKBP12 [6]. FKBP38
binds to mTOR and inhibits its activity. Rheb interacts directly with FKBP38 and prevents
its association with mTOR in a GTP–dependent manner. Moreover, FKBP38 bound to GTP-γ-S,
a nonhydrolyzable GTP analogon, has a much higher binding affinity for Rheb than the
GDP-bound form [6]. However, two other publications re-evaluated the results and came
to different conclusions. The first study confirmed the interaction between Rheb and
FKBP38 but disagreed, that the bound nucleotide has an effect on the interaction [7].
The second study contradicted both studies, since they could not detect any interaction
between Rheb and FKBP38 [8]. To clarify whether there is an interaction and if it
is nucleotide dependent, NMR monitored interaction studies were performed employing
a C-terminal truncated construct of human Rheb (1-170 = RhebΔCT) that cannot be farnesylated
and the biochemically defined binding region on FKPB38 (FKBP12-like = FKBP38-BD).
Based on our data RhebΔCT –GDP does not significantly interact with FKBP38-BP. 15N-FKBP38-BD
titrated with a 9-mer peptide corresponding to the Rheb switch 1 did also not result
spectral changes. Thus the proposed importance of the switch 1 region for the interaction
with FKBP38 maybe indirect by influencing the nucleotide binding. However, we observed
a weak interaction between RhebΔCT bound to a GTPanalogon (GppNHp) and FKBP38-BD.
Mapping of the observed spectral changes on the structure of Rheb-GTP suggests that
FKBP38 targets the switch 2 region, loop ∼109-112 and the neighboring b-sheet region.
We further analyzed the backbone dynamics of RhebΔCT –GDP and –GppNHp using 15N relaxtion
data (T1, T2 and heteronuclear NOE). Based on these data the phosphorylation loop,
the switch regions and the loop around residues ∼109-112 show increased backbone dynamics
that modulated by the nucleotide binding. These increased dynamics may allow Rheb
to interact with several different interaction partners such as FKBP38.
[1] Y. Li, M.N. Corradetti, K. Inoki, K.L. Guan, Trends Biochem. Sci. 29 (2004) 32–38.
[2] B.D. Manning, L.C. Cantley, Trends Biochem. Sci. 28 (2003) 573–576.
[3] D.J. Kwiatkowski, Cancer Biol. Ther. 2 (2003) 471– 476.
[4] Buerger, C., B. DeVries, and V. Stambolic. Biochem. and Bioph. Res. Comm., 2006.
344(3): p. 869-880.
[5] Groenewoud, M.J. and F.J. Zwartkruis. Biochem Soc Trans, 2013. 41(4): p. 951-5.
[6] Bai, X., et al. Science, 2007. 318: p. 977-980.
[7] Wang, X., et al.J Biol Chem, 2008. 283(45): p. 30482-92. [8] Uhlenbrock, K., et
al., FEBS Lett, 2009. 583(6): p. 965-70
PL-078
Unraveling the nature of TDP-43 aggregates from its putative aggregation domain
Miguel Mompeán1, Rubén Hervás2, Yunyao Xu3, Timothy H. Tran4, Emanuele Buratti5, Francisco
Baralle5, Liang Tong4, Mariano Carrión-Vázquez2, Ann E. McDermott3, Douglas V Laurents1
1Instituto Química Física Rocasolano, 2Instituto Cajal, IC-CSIC, 3Department of Chemistry,
Columbia University, 4Department of Biological Sciences, Columbia University, 5International
Centre for Genetic Engineering and Biotechnology
TDP-43 is an RNA processing protein that can form inclusions of debatable nature implicated
in neurodegenerative diseases. Within the putative aggregation domain, repeats of
residues 341-366 can recruit endogenous TDP-43 into aggregates inside cells1. Recently,
we showed that a coil to β-hairpin transition in a short peptide corresponding to
TDP-43 residues 341-357 enables oligomerization2. We have used a broad battery of
biophysical experiments, including chromophore and antibody binding, electron microscopy
(EM), circular dichroism (CD), solution and solid-state NMR, and X-ray to shed light
on the nature of these aggregates. Based on these findings, structural models for
TDP-43(341-357) oligomers have been constructed, refined, verified, and analyzed using
computational methods, ranging from Docking and Molecular Dynamics simulations to
Semiempirical Quantum Mechanics calculations. Interestingly, TDP-43(341-357) β-hairpins
assemble into a novel parallel β-turn configuration showing cross-β spine, cooperative
H-bonding and tight side chain packing3. These results expand the amyloid foldome
and could guide rational drug design to prevent this process.
REFERENCES:
1. Budini M, Buratti E, Stuani C, Guarnaccia C, Romano V, De Conti L, Baralle FE.
Cellular Model of TAR DNA-Binding Protein 43(TDP-43) Aggregation Based on Its C-Terminal
Gln/Asn-Rich Region. J. Biol. Chem. 2012, 287:7512-7525
2. Structural Characterization of the Minimal Segment of TDP-43 Competent for Aggregation.
Mompeán M, Buratti E, Guarnaccia C, Brito RM, Chakrabartty A, Baralle FE, Laurents
DV. Arch. Biochem. Biophys. 2014, 545:53-62.
3. Structural Evidence of Amyloid Fibril Formation in the Putative Aggregation Domain
of TDP-43. Mompeán M, Hervás R, Xu Y, Tran TH, Guarnaccia C, Buratti E, Baralle FE,
Tong L, Carrión-Vázquez M, McDermott AE, Laurents DV. J. Phys. Chem. Lett. 2015, 6:2608-2’06:15.
PL-079
The role of the structural NADP+ binding site in human glucose 6-phosphate dehydrogenase
Mona Alonazi1,2, Paul Engel1
1School of Biomolecular and Biomedical Science, Conway Institute, UCD., 2King Saud
University, Sciences, Biochemistry department.
The crystal structure of a human glucose 6-phosphate dehydrogenase (G6PD) shows that
each subunit has two NADP+ sites; in addition to a catalytic site there is a “structural”
site which is distant from the catalytic coenzyme site. Mutations causing severe deficiency
tend to cluster round and close to the dimer interface and the structural NADP+, indicating
that the integrity of these areas is important for enzyme stability and therefore
for maintenance of activity. In order to understand the molecular basis of G6PD deficiency,
and to have a clearer indication about the role of some features of the three-dimensional
structure, a fuller study of the second, “structural” NADP+ binding site is needed.
Human G6PD controls the first committed step in the pentose phosphate pathway. It
catalyses the oxidation of glucose 6-phosphate to gluconolactone 6-phosphate, generating
NADPH which is essential, amongst other things, for protection against oxidative stress.
The human enzyme can be active in dimer or tetramer forms. Human G6PD of “structural”
NADP+ per subunit of enzyme. This tightly-bound NADP+ can be reduced by G6P, probably
following migration to the catalytic site. The importance of NADP+ for stability is
explained by the structural NADP+ site, which is not conserved in prokaryotes. After
removing the tightly bound “structural” NADP+ the enzyme is still active but not stable.
The effects of different NADP+ fragments on the stability of human recombinant G6PD
have been investigated. NADP+ is crucial for the long term stability of human G6PD,
and only one of NADP+ analogues which is adenosine diphosphate ribose - 2’-phosphate
was able to slightly promote the stability of enzyme.
PL-080
Molecular characterization of specific positively selected sites in mammalian visual
pigment evolution
Miguel A. Fernández-Sampedro1, Eva Ramon1, Brandon M. Invergo2, Jaume Bertranpetit2,
Pere Garriga1
1Grup de Biotecnologia Molecular i Industrial., 2IBE – Institute of Evolutionary Biology
Visual rhodopsin is a member of the G-protein coupled receptors superfamily. This
membrane protein consists of a 11-cis-retinal cromophore bound to a seven transmembrane
protein, opsin, by means of a protonated Schiff base linkage. It has an important
role as a dim light photoreceptor in the retina of the eye. By statistical models,
where episodic selection in rhodopsin is tested on one branch of the phylogeny against
a background of neutral or purifying selection on the rest of the tree, we have found
some significant evidence of specific positively selected sites in early mammalian
divergence. We have chosen the three amino acid sites identified with the highest
posterior probability of having been targets of positive selection to perform experimental
studies, i.e. 13 (positively selected from M to F), 225 (positively selected from
R to Q) and 346 (positively selected from S to A). We have constructed, expressed,
immunopurified and functionally characterized the proposed candidates, F13M, Q225R
and A346S rhodopsin mutants located at the N-terminus, the transmembrane domain and
the C-terminus region of the protein respectively. From the analysis of the molecular
features of the F13M mutant, we conclude that position 13 is very important for protein
folding and also for proper protein glycosylation, since we only could observe cromophore
regeneration after its rescue in the double cysteine (N2C/D282C) mutant background
that stabilizes the N-terminal extracellular domain of the protein. Our results also
show that mutants Q225R and A346S alter the G-protein activation rate, and hydroxylamine
susceptibility in the dark-adapted state. In the case of Q225R, disrupting critical
interactions with the neighbouring Y136 of the conserved D/ERY motif, critical in
Gt activation, could cause the lower Gt activation ability. The mutant A346S would
create a potential additional phosphorylation site in the protein which could affect
rhodopsin phosphorylation after photoactivation and, in turn, could affect the binding
affinity of arrestin, a regulator of rhodopsin deactivation. This extra phosphorylation
site could provide an evolutionary explanation for the enhanced response observed
in the case of Gt activation. In conclusion, these results highlight the importance
of molecular investigations of positive selected sites in rhodopsin evolution and
the relevance of structural and functional analysis of these sites in unravelling
the molecular basis of visual pigment evolution.
PL-081
Natural evolution sheds light on modern drug resistance in protein kinases
Marc Hoemberger1, Christopher Wilson1, Roman Agafonov1, Dorothee Kern1
1HHMI & Department of Biochemistry, Brandeis University
The anti-cancer drug imatinib exhibits highly specific binding to the human kinase
and oncogene Abl with a three thousand fold weaker affinity for the structurally and
functionally very similar kinase Src. It has been shown recently that the major difference
in binding of imatinib to Abl and Src stems from an induced fit after binding of the
drug. To further understand the mechanism of imatinib binding to its target we used
ancestral sequence reconstruction (ASR) and resurrected enzymes along the node from
the common ancestor of Abl and Src up to the extant kinases. We show that imatinib
affinity is gained towards the evolution of extant Abl while it is lost towards evolving
Src. The combination of ASR and crystallographic data of the ancestors in addition
to kinetics data allowed us to identify a subset of residues involved in imatinib
specificity sufficient to switch from an intermediate binder to a tight binder. Preliminary
data shows that a network of hydrogen bonds and packing interactions stabilize the
kinked p-loop conformation for tight binders thus allowing for more interactions between
the kinase and the drug. Strikingly, many of these residues were identified in human
cancer patients as “hot spots” for the development of resistance mutations. Further
investigation into the identified subset of residues in combination with these commonly
found imatinib resistance mutations will allow us to understand emerging drug resistances
better.
PL-082
An evolutionary view of the cold adapted catalysis of enzymes
Vy Nguyen1, Christopher Wilson1, Dorothee Kern1
1HHMI & Department of Biochemistry, Brandeis University
The diversity in protein function that we see today arose as a result of life adapting
to a cooling earth. How did enzymes, the catalysts of many crucial cellular processes,
achieve this cold adaptation? This is a challenging question to answer because ancient
sequences of proteins that existed billions of years ago are not available. To address
this question we used ancestral sequence reconstruction to create adenylate kinase
(Adk) enzymes from the divergence of Anaerobic and Aerobic Firmicutes towards modern
day thermophilic, mesophilic and psychrophilic organisms. Adk is a phosphotransferase
that catalyzes the conversion of two ADP molecules into ATP and AMP. We make the following
observations. First, all ancestral enzymes are active with optimal catalytic rates
linearly corresponding to the temperature of the environments where these proteins
would have been found. Most strikingly, the catalytic rate of our oldest Adk ancestor
exhibits a higher enthalpy of activation at low temperatures as compared to the modern
thermophilic Adk. This suggests a large enthalpic penalty had to be paid for reactions
to occur at cold temperatures in an ancestor that existed in a hot environment. Second,
several high resolution crystal structures of extant proteins that we solved (1.2Å
– 1.6Å), show that the oldest ancestors were more rigid than the modern Adks due to
an intricate salt-bridge network. This work, thus shows for the first time, the molecular
and thermodynamic determinants of cold adaptation in an enzyme over a time period
that spans billions of years.
PL-083
Induced oxidative modification of plasma and cellular fibrin-stabilizing factor
Anna Bychkova1, Tatiana Danilova1, Alexander Shchegolikhin1, Vera Leonova1, Marina
Biryukova1, Elizaveta Kostanova1, Alexey Kononikhin0, Anna Bugrova1, Evgeny Nikolaev0,
Mark Rosenfeld1
1N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 2Institute
for Energy Problems of Chemical Physics, Russian Academy of Sciences
The main function of plasma fibrin-stabilizing factor pFXIII is to catalyze the formation
of the intermolecular covalent cross-links between both γand αfibrin polypeptide chains.
The crosslinking crucially affects mechanical strength of fibrin and its resistance
against fibrinolysis. The precise role of cellular fibrin-stabilizing factor cFXIII
remains poorly understood. pFXIII is a heterotetramer (FXIII-A2B2) consisting of two
single-stranded catalytic A subunits (FXIII-A2), and two identical single-stranded
inhibitory/carrier B subunits (FXIII-B2). The subunits are held together by weak non-covalent
bonds. Contrary to plasma FXIII, cFXIII is a dimer (FXIII-A2) devoid of B subunits.
As well as many other proteins circulating in the bloodstream, pFXIII is known to
be a target for reactive oxygen species (ROS) causing processes of protein oxidative
modification. Since the conversion of pFXIII to the active form of the enzyme (FXIIIа)
is a multistage process, ozone-induced oxidation of pFXIII has been investigated at
different stages of its enzyme activation. The biochemical results point to an inhibition
of enzymatic FXIIIа activity depending largely on the stage of the pFXIII conversion
into FXIIIа at which oxidation was carried out. UV-, FTIR- and Raman spectroscopy
demonstrated that chemical transformation of cyclic, NH, SH and S–S groups mainly
determines the oxidation of amino acid residues of pFXIII polypeptide chains. Conversion
of pFXIII to FXIIIa proved to increase protein susceptibility to oxidation in the
order: pFXIII < pFXIII activated by thrombin < pFXIII in the presence of calcium ions < FXIIIa.
With the aid of mass-spectrometry it has been demonstrated that oxidation leads to
decreasing FXIII-A and FXIII-B coverage both in the forms of zymogen and in the presence
of calcium ions. A group of amino acid residues involved in oxidation modification
of pFXIII is identified in this study. The oxidation of either cFXIII or cFXIIIa has
revealed an almost complete loss of enzyme activity caused by dramatic changes in
the primary and secondary structure of the proteins detected by the FTIR data. Taking
into account these new findings, it seems reasonable to assume that the inhibitory/carrier
FXIII-B subunits can serve as scavengers of ROS. Hypothetically, this mechanism could
help to protect the key amino acid residues of the FXIII-A subunits responsible for
the enzymatic function of FXIIIa.
The study was supported by RFBR, research project No. 15-15-04-08188a. Mass spectrometry
study was supported by the Russian Scientific Foundation grant No. 14-24-00114.
PL-084
Performance and quality. making microcalorimetry simple with microcal peaq-itc
Natalia Markova1, Ronan O’Brien1, Mark Arsenault1
1MicroCal, Malvern Instruments Ltd.
Dynamic interactions involving biomolecules drive and regulate all biological processes.
Studies of biomolecular interactions are fundamentally important in all areas of life
sciences. Data provided by Isothermal Titration Calorimetry (ITC) enables scientists
in academia and industry to directly and quantitatively characterize these interactions
in solution. MicroCal PEAQ-ITC, the latest generation of MicroCal ITC instrumentation,
offers a whole range of solutions for addressing current bottlenecks associated with
interaction analysis. Among the most recognized challenges are the needs to adequately
address a broad range of binding affinities and to reliably interpret binding data
complicated by the presence of inactive protein fraction or inherent uncertainty in
the concentration of a ligand. Consistently high performance of MicroCal PEAQ-ITC
enables increased confidence and data resolution when measuring low heats at low or
uncertain sample concentrations and complex binding modes. The new MicroCal PEAQ-ITC
analysis software allows for utomated data analysis, minimizing analysis time and
user subjectivity in assessing data quality. Data quality is determined and advanced
fitting performed in a few seconds per experiment allowing for analysis of large data
sets of 50 or more experiments in a matter of seconds.
PL-085
Glutamine-rich activation domain of transcription factor Sp1 - biochemical activity
and structure
Jun Kuwahara1, Chisana Uwatoko1, Emi Hibino2, Katsumi Matsuzaki2, Masaru Hoshino2
1Faculty of Pharmaceutical Sciences, Doshisha Women’s University, 2Graduate School
of Pharmaceutical Sciences, Kyoto University
Transcription factor Sp1 is ubiquitously expressed in a mammalian cell, activates
reasonably large subset of mammalian genes, and is involved in the early development
of an organism. The protein comprises two glutamine-rich (Q-rich) regions (A and B
domains) located in its N-terminal half, while three tandem repeats of C2H2 zinc finger
motif at its C-terminus binds directly to a GC-rich element (GC box) of DNA. In general,
Q-rich domain is one of the typical motifs found in trans-activation domain of transcription
factors together with acidic and proline-rich domains. Transcriptional signal of Sp1
are transmitted via interaction between Q-rich domains of Sp1 and different classes
of nuclear proteins, such as TATA-binding protein (TBP) associated factors (TAFs)
in components of basic transcription factor complexes (TFII). In addition, self-association
of Sp1 via Q-rich domains is also important for its regulation of transcriptional
activity. It has been considered that an Sp1! molecule bound to a ’distal’ GC-box
synergistically interacts with another Sp1 molecule at a ’proximal’ binding site.
Although formation of multimers via Q-rich domains seems functionally important for
Sp1, little is known about relevance between biological activity and structural nature
of Q-rich domains. We analyzed nature of glutamine-rich domains of Sp1 by biochemical
and physicochemical methods. We found that Q-rich domains do not have clear secondary
structure whereas they can indicate biochemical activity. Detailed analysis of NMR
spectra indicated interaction between the domains. The Q-rich domains of Sp1 might
be one of the intrinsically disordered proteins (IDP).
PL-086
CHIPping away at the yeast proteome: redesigning an E3 ubiquitin ligase for targeted
protein degradation
Michael Hinrichsen1, Lynne Regan1
1Yale University
One of the central goals of synthetic biology is to exploit biological systems in
order to produce compounds of therapeutic or industrial value 1. Often, these efforts
are complicated by the many natural biochemical pathways in cells that can compete
for the same small molecule precursors. Currently, the most common solution is to
simply delete the genes coding for the competing enzymes 2. While such an approach
has been successful, it is only applicable to nonessential genes and can produce unintended
off-target effects such as decreased cell viability 2. An alternative strategy is
to instead target proteins directly for degradation. Using this strategy, scientists
would first grow cultures of engineered cells to high densities under permissive conditions
(i.e. targeted proteins are stably expressed). Then, once sufficient cell density
has been reached, enzymes of competing pathways would be rapidly degraded, resulting
in the rapid production of high concentrations of the compound of interest. We propose
to create such a tool by reengineering the C-terminus of Hsp70 interacting protein
(CHIP), an E3 ubiquitin ligase. CHIP recognizes substrate proteins through a short
C-terminal peptide tag on target proteins3. We have shown that fusing this tag to
non-native substrates is sufficient for ubiquitination in vitro (data not published).
Cellular assays have also been performed in S. Cerevisiae, a model organism commonly
used in metabolic engineering applications1. As a number of native yeast proteins
possess C-termini similar to that of CHIP’s native substrates (data not published),
it was necessary to develop an orthogonal CHIP-peptide pair. This was achieved by
replacing CHIP’s natural TPR ligand-binding domain with a ligand-binding domain engineered
previously in the Regan Lab 4. The altered CHIP construct has been shown to be active
both in vitro and in vivo, and produces an altered growth phenotype when targeted
against an enzyme involved in uracil biosynthesis. Future work will focus on further
kinetic characterization of the engineered enzyme, increasing its activity, and introducing
the system into a proof of concept synthetic biology application.
PM-001
Functional characterization of proteins by domain architecture
Roxanne Yamashita1, Lewis Geer1, Lianyi Han1, Lanczycki Christopher1, Shennan Lu1,
Jane He1, Josie Wang1, CDD Curation Team1, Aron Marchler-Bauer1
1National Institutes of Health/National Center for Biotechnology Information
Advances in modern sequencing techniques have resulted in an explosion of genomic
data. Correctly classifying this new wealth of information can be daunting not only
because of the sheer volume of sequence data, but also because the propagation of
erroneous and less-than-ideal names and functional characterizations in the current
databases gets in the way of functional classification by mere sequence similarity.
We are investigating the extent to which protein domain architecture can be utilized
to define groups of proteins with similarities in molecular function, and whether
we can derive corresponding functional “labels”, starting with some of the most common
domain architectures found in bacteria. To this end, we have developed an in-house
procedure called SPARCLE (’SPecific ARChitecture Labeling Engine’) that lets us track
and examine specific or sub-family domain architectures, resulting from annotating
protein sequences with domain footprints provided by the Conserved Domain Database
(CDD), which includes hierarchical classifications for many common domain families.
We will discuss how the proteins are grouped into specific architectures, our successes
in assigning functional labels, and the major limitations we have encountered to date.
While we will be able to assign functional labels to a large fraction of protein models
derived from genome sequences, this effort has the added benefit of pointing out insufficient
coverage and resolution of the current protein domain model collections that constitute
CDD. We will also discuss alternative procedures that utilize pre-computed domain
annotation for clustering protein sequences at a level that is well suited for functional
labeling. We hope that this preliminary study will help to identify approaches that
facilitate rapid and accurate annotation of genomes with a minimum of manual intervention.