Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis
| Autor: | Peter J. Bond, Pedro M. Alzari, Alexandra East, Ariel E. Mechaly, Alejandro Buschiazzo, Nathalie Nadeau, Olivera Francetic, Gerard H. M. Huysmans, Diana Tello-Manigne, Anthony P. Pugsley, Cédric Bernarde |
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| Přispěvatelé: | Department of Chemistry [Cambridge, UK], University of Cambridge [UK] (CAM), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Systèmes macromoléculaires et Signalisation, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Bioinformatics Institute [Singapore], Agency for science, technology and research [Singapore] (A*STAR), A.E. was supported by an EPSRC PhD studentship. A.E.M. was recipient of a DK fellowship from the Basque Government. G.H.M.H. was a recipient of a Marie Curie Intra-European Fellowship (PIEF-GA-2010-272611) and an EMBO-Pasteur fellowship (ALTF 1088–2010)., We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and its staff for assistance in using beamline ID14.2. We thank Ingrid Guilvout for help with the PulA size-exclusion chromatography and Jacques D'Alayer for N-terminal sequencing of PulA. This study was supported by the ANR BLANC grant 1531-02. The computational work benefited from use of the Darwin Supercomputer of the University of Cambridge High Performance Computing Service (http://www.hpc.cam.ac.uk/), provided by Dell Inc. using Strategic Research, Infrastructure Funding from the Higher Education Funding Council for England, the Swiss National Supercomputing Center, via DECI/PRACE-2IP, and the HECToR supercomputing service via the UK High-End Computing Consortium for Biomolecular Simulation/EPSRC., We thank V. Shevchik and X. Robert, members of the Molecular Genetics Unit, Structural Microbiology Unit and Laboratory of Macromolecular Systems Signaling for help and support. We are grateful to Evelyne Richet for the critical reading of the manuscript., European Project: 272611,EC:FP7:PEOPLE,FP7-PEOPLE-2010-IEF,FAPUL(2011), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
[SDV]Life Sciences [q-bio] Lipid Bilayers POPE MESH: Amino Acid Sequence Crystallography X-Ray Molecular dynamics Structural Biology Klebsiella MESH: Molecular Dynamics Simulation MESH: Bacterial Proteins MESH: Exocytosis peripheral membrane proteins MESH: Lipid Bilayers Peripheral membrane protein MESH: Lipoproteins lipoprotein sorting Membrane Biochemistry MESH: Membrane Proteins Protein Binding liposomes Glycoside Hydrolases Lipoproteins 030106 microbiology Molecular Sequence Data Biology Molecular Dynamics Simulation Exocytosis 03 medical and health sciences Bacterial Proteins Hydrolase MESH: Glycoside Hydrolases Inner membrane MESH: Protein Binding Secretion Amino Acid Sequence Molecular Biology X-ray crystallography pullulanase MESH: Molecular Sequence Data Binding Sites Pullulanase Cell Membrane type II secretion Membrane Proteins Periplasmic space MESH: Crystallography X-Ray molecular dynamics MESH: Klebsiella MESH: Binding Sites secretion signal MESH: Cell Membrane |
| Zdroj: | Structure Structure, Elsevier (Cell Press), 2016, 24 (1), pp.92-104. ⟨10.1016/j.str.2015.10.023⟩ Structure, 2016, 24 (1), pp.92-104. ⟨10.1016/j.str.2015.10.023⟩ |
| ISSN: | 1878-4186 0969-2126 |
| DOI: | 10.1016/j.str.2015.10.023⟩ |
| Popis: | International audience; The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain. |
| Databáze: | OpenAIRE |
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