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      Conformational Response to Ligand Binding in Phosphomannomutase2 : INSIGHTS INTO INBORN GLYCOSYLATION DISORDER *

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          Background: Mutations in phosphomannomutase2 cause glycosylation disorder, a disease without a cure that will largely benefit from accurate ligand-bound models.

          Results: We obtained two models of phospomannomutase2 bound to glucose 1,6-bisphosphate and validated them with limited proteolysis.

          Conclusion: Ligand binding induces a large conformational transition in PMM2.

          Significance: We produce and validate closed-form models of PMM2 that represent a starting point for rational drug discovery.


          The most common glycosylation disorder is caused by mutations in the gene encoding phosphomannomutase2, producing a disease still without a cure. Phosphomannomutase2, a homodimer in which each chain is composed of two domains, requires a bisphosphate sugar (either mannose or glucose) as activator, opening a possible drug design path for therapeutic purposes. The crystal structure of human phosphomannomutase2, however, lacks bound substrate and a key active site loop. To speed up drug discovery, we present here the first structural model of a bisphosphate substrate bound to human phosphomannomutase2. Taking advantage of recent developments in all-atom simulation techniques in combination with limited and site-directed proteolysis, we demonstrated that α-glucose 1,6-bisphosphate can adopt two low energy orientations as required for catalysis. Upon ligand binding, the two domains come close, making the protein more compact, in analogy to the enzyme in the crystals from Leishmania mexicana. Moreover, proteolysis was also carried out on two common mutants, R141H and F119L. It was an unexpected finding that the mutant most frequently found in patients, R141H, although inactive, does bind α-glucose 1,6-bisphosphate and changes conformation.

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          Most cited references 23

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          JOY: protein sequence-structure representation and analysis.

          JOY is a program to annotate protein sequence alignments with three-dimensional (3D) structural features. It was developed to display 3D structural information in a sequence alignment and to help understand the conservation of amino acids in their specific local environments. : The JOY representation now constitutes an essential part of the two databases of protein structure alignments: HOMSTRAD ( ) and CAMPASS ( uk/campass). It has also been successfully used for identifying distant evolutionary relationships. The program can be obtained via anonymous ftp from from the directory /pub/joy/. The address for the JOY server is
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            Successful prenatal mannose treatment for congenital disorder of glycosylation-Ia in mice.

            Congenital disorder of glycosylation-Ia (CDG-Ia, also known as PMM2-CDG) is caused by mutations in the gene that encodes phosphomannomutase 2 (PMM2, EC leading to a multisystemic disease with severe psychomotor and mental retardation. In a hypomorphic Pmm2 mouse model, we were able to overcome embryonic lethality by feeding mannose to pregnant dams. The results underline the essential role of glycosylation in embryonic development and may open new treatment options for this disease.
              • Record: found
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              Is Open Access

              Secondary structure assignment that accurately reflects physical and evolutionary characteristics

              Background Secondary structure is used in hierarchical classification of protein structures, identification of protein features, such as helix caps and loops, for fold recognition, and as a precursor to ab initio structure prediction. There are several methods available for assigning secondary structure if the three-dimensional structure of the protein is known. Unfortunately they differ in their definitions, particularly in the exact positions of the termini. Additionally, most existing methods rely on hydrogen bonding, which means that important secondary structural classes, such as isolated β-strands and poly-proline helices cannot be identified as they do not have characteristic hydrogen-bonding patterns. For this reason we have developed a more accurate method for assigning secondary structure based on main chain geometry, which also allows a more comprehensive assignment of secondary structure. Results We define secondary structure based on a number of geometric parameters. Helices are defined based on whether they fit inside an imaginary cylinder: residues must be within the correct radius of a central axis. Different types of helices (alpha, 310 or π) are assigned on the basis of the angle between successive peptide bonds. β-strands are assigned based on backbone dihedrals and with alternating peptide bonds. Thus hydrogen bonding is not required and β-strands can be within a parallel sheet, antiparallel sheet, or can be isolated. Poly-proline helices are defined similarly, although with three-fold symmetry. Conclusion We find that our method better assigns secondary structure than existing methods. Specifically, we find that comparing our methods with those of others, amino-acid trends at helix caps are stronger, secondary structural elements less likely to be concatenated together and secondary structure guided sequence alignment is improved. We conclude, therefore, that secondary structure assignments using our method better reflects physical and evolutionary characteristics of proteins. The program is available from

                Author and article information

                J Biol Chem
                J. Biol. Chem
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                12 December 2014
                16 October 2014
                16 October 2014
                : 289
                : 50
                : 34900-34910
                From the []Istituto di Chimica Biomolecolare-Consiglio Nazionale Delle Ricerche, 80078 Pozzuoli, Italy,
                [§ ]Joint Barcelona Supercomputing Center-Center for Genomic Regulation-Institute for Research in Biomedicine Research Program in Computational Biology, Barcelona Supercomputing Center, c/Jordi Girona 29, 08034 Barcelona, Spain,
                []Dipartimento di Farmacia, Università degli Studi di Salerno, 84084 Fisciano, Italy,
                [** ]Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys 23, 08010 Barcelona, Spain, and
                []Dipartimento di Biologia, Università Federico II, 80126 Naples, Italy
                Author notes
                [2 ] To whom correspondence may be addressed: LifeScience Dept., Barcelona Supercomputing Center, c/Jordi Girona 29, 08034 Barcelona, Spain. Tel.: 34-934137727; Fax: 34-934137721; E-mail: victor.guallar@ .
                [3 ] To whom correspondence may be addressed: Dipartimento di Biologia, Complesso di Monte Sant'Angelo, Via Cinthia, Naples 80126, Italy. Tel.: 39-081-679-118; Fax: 39-081-679233; E-mail: cubellis@ .

                Both authors contributed equally to this work.

                © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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                Molecular Biophysics


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