High-resolution mass spectrometry of small molecules bound to membrane proteins

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Abstract

Small molecules are known to stabilise membrane proteins and to modulate function and oligomeric state, but their identity is often hard to define. Here we develop and apply a high-resolution, Orbitrap mass spectrometer for intact membrane protein-ligand complexes. Using this platform we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.

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Most cited references 28

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A rapid method of total lipid extraction and purification.

E G BLIGH, W. Dyer (1959)

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Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles.

Michael T. Marty, Andrew J Baldwin, Erik Marklund … (2015)

Interpretation of mass spectra is challenging because they report a ratio of two physical quantities, mass and charge, which may each have multiple components that overlap in m/z. Previous approaches to disentangling the two have focused on peak assignment or fitting. However, the former struggle with complex spectra, and the latter are generally computationally intensive and may require substantial manual intervention. We propose a new data analysis approach that employs a Bayesian framework to separate the mass and charge dimensions. On the basis of this approach, we developed UniDec (Universal Deconvolution), software that provides a rapid, robust, and flexible deconvolution of mass spectra and ion mobility-mass spectra with minimal user intervention. Incorporation of the charge-state distribution in the Bayesian prior probabilities provides separation of the m/z spectrum into its physical mass and charge components. We have evaluated our approach using systems of increasing complexity, enabling us to deduce lipid binding to membrane proteins, to probe the dynamics of subunit exchange reactions, and to characterize polydispersity in both protein assemblies and lipoprotein Nanodiscs. The general utility of our approach will greatly facilitate analysis of ion mobility and mass spectra.

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Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex.

Qiuxiang Tan, Ya Zhu, Jian LI … (2013)

The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom-resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor-gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.

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Author and article information

Journal

Journal ID (nlm-journal-id): 101215604

Journal ID (pubmed-jr-id): 32338

Journal ID (nlm-ta): Nat Methods

Journal ID (iso-abbrev): Nat. Methods

Title: Nature methods

ISSN (Print): 1548-7091

ISSN (Electronic): 1548-7105

Publication date Nihms-submitted: 29 January 2016

Publication date (Electronic): 22 February 2016

Publication date (Print): April 2016

Publication date PMC-release: 01 October 2016

Volume: 13

Issue: 4

Pages: 333-336

Affiliations

[1 ]Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK

[2 ]Department of Biochemistry, University of Oxford, Oxford, UK

[3 ]CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Pudong, China

[4 ]Thermo Fisher Scientific, Bremen, Germany

Author notes

Corresponding author Correspondence to: Carol V Robinson - carol.robinson@ 123456chem.ox.ac.uk

[5]

Present Address UMR 5235 CNRS, Université de Montpellier, Montpellier, France

Author contributions

J.G. and C.V.R., with assistance from J.T.S.H., designed the research. M.B., E.D., A.M., T.M. and J.G. modified the Q Exactive mass spectrometer and optimised the MS experiment for membrane protein complexes. J.G., J.A.C.D., I.L., K.G., J.T.S.H. and W.S. expressed and purified membrane proteins in appropriate conditions for non-denaturing MS. J.G., J.A.C.D. and I.L. performed MS experiments. J.G. and J.A.C.D. performed lipidomics experiments and data analysis. Y.Z. and B.W. provided sample of the CCR5 protein. N.G.H. and C.K. provided sample of the OmpF protein and OBS1 peptide. C.B. set up the lipidomics platform. M.T.M. modified the Unidec software for use in this work. J.G. and C.V.R. wrote the manuscript with contributions from all other authors.

Article

Manuscript ID: EMS66983

DOI: 10.1038/nmeth.3771

PMC ID: 4856209

PubMed ID: 26901650

SO-VID: e62bb5ad-17a2-4cfa-ade2-7623375aa621

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High-resolution mass spectrometry of small molecules bound to membrane proteins

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Abstract

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Cancer Immunotherapy

Most cited references 28

A rapid method of total lipid extraction and purification.

Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles.

Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex.

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