title: Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5.
abstract: A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD-Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, 48160 Derio, Spain;
authors.forename: María
authors.initials: M
authors.lastname: Lucas
authors.affiliation: Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Gaspar
authors.affiliation: Joint Barcelona Supercomputing Center-Institute for Research in Biomedicine Research Program in Computational Biology, Barcelona Supercomputing Center, 08034 Barcelona, Spain; and.
authors.forename: Chiara
authors.initials: C
authors.lastname: Pallara
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, 48160 Derio, Spain;
authors.forename: Adriana Lucely
authors.initials: AL
authors.lastname: Rojas
authors.affiliation: Joint Barcelona Supercomputing Center-Institute for Research in Biomedicine Research Program in Computational Biology, Barcelona Supercomputing Center, 08034 Barcelona, Spain; and.
authors.forename: Juan
authors.initials: J
authors.lastname: Fernández-Recio
authors.affiliation: Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; machnerm@mail.nih.gov ahierro@cicbiogune.es.
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Machner
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, 48160 Derio, Spain; IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain machnerm@mail.nih.gov ahierro@cicbiogune.es.
authors.forename: Aitor
authors.initials: A
authors.lastname: Hierro
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D011992:Endosomes / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D016952:Legionella pneumophila / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D054466:Phospholipases A1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D054466:Phospholipases A1; D020696:rab5 GTP-Binding Proteins
keywords: X-ray crystallography; allosteric modulation; membrane composition; pathogenic bacteria
references.citation: J Exp Med. 1983 Dec 1;158(6):2108-26
references.pmid: 6644240
references.citation: Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9431-6
references.pmid: 10920208
references.citation: Infect Immun. 2006 Jun;74(6):3597-606
references.pmid: 16714592
references.citation: Cell. 2010 Nov 24;143(5):694-702
references.pmid: 21111231
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W368-71
references.pmid: 15980491
references.citation: PLoS Pathog. 2012;8(12):e1003082
references.pmid: 23271971
references.citation: J Mol Biol. 2004 Nov 12;344(1):71-89
references.pmid: 15504403
references.citation: J Biol Chem. 2002 Jan 18;277(3):2266-74
references.pmid: 11689574
references.citation: J Biol Chem. 2005 Nov 4;280(44):36773-83
references.pmid: 16103116
references.citation: Trends Microbiol. 2012 Jun;20(6):299-306
references.pmid: 22494803
references.citation: Curr Opin Microbiol. 2009 Feb;12(1):67-73
references.pmid: 19157961
references.citation: Sci Signal. 2009 Nov 03;2(95):ra71
references.pmid: 19887681
references.citation: Nature. 2005 Jul 21;436(7049):415-9
references.pmid: 16034420
references.citation: J Biol Chem. 2012 Aug 24;287(35):29654-63
references.pmid: 22740689
references.citation: Science. 2009 Jan 9;323(5911):266-8
references.pmid: 19131631
references.citation: Nat Struct Mol Biol. 2004 Oct;11(10):975-83
references.pmid: 15378032
references.citation: Proteins. 2003 Feb 15;50(3):437-50
references.pmid: 12557186
references.citation: Nature. 1991 Jun 6;351(6326):491-4
references.pmid: 2046751
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 29;102(13):4866-71
references.pmid: 15781869
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):133-44
references.pmid: 20124693
references.citation: PLoS Pathog. 2012;8(4):e1002637
references.pmid: 22496657
references.citation: J Bacteriol. 2001 Jul;183(14):4330-44
references.pmid: 11418575
references.citation: Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):10866-71
references.pmid: 20534488
references.citation: Nucleic Acids Res. 2014 Jan;42(Database issue):D222-30
references.pmid: 24288371
references.citation: Chem Rev. 2011 Oct 12;111(10):6130-85
references.pmid: 21910409
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Future Microbiol. 2012 Feb;7(2):241-57
references.pmid: 22324993
references.citation: Nature. 1993 Apr 29;362(6423):814-20
references.pmid: 8479519
references.citation: Protein Expr Purif. 1999 Feb;15(1):34-9
references.pmid: 10024467
references.citation: J Biol Chem. 2001 Apr 27;276(17):13982-8
references.pmid: 11278565
references.citation: Structure. 2010 Nov 10;18(11):1399-409
references.pmid: 21070939
references.citation: Biophys J. 2001 Jun;80(6):2946-53
references.pmid: 11371467
references.citation: Mol Microbiol. 2011 Dec;82(6):1454-67
references.pmid: 22040088
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: Cell. 1999 Apr 30;97(3):349-60
references.pmid: 10319815
references.citation: Biochim Biophys Acta. 2006 Sep;1761(9):995-1013
references.pmid: 16931141
references.citation: Cell Host Microbe. 2008 Nov 13;4(5):434-46
references.pmid: 18996344
references.citation: Biochim Biophys Acta. 2014 Aug;1843(8):1578-91
references.pmid: 24389247
references.citation: J Bacteriol. 2005 Nov;187(22):7716-26
references.pmid: 16267296
references.citation: Cell Microbiol. 2013 Nov;15(11):1783-95
references.pmid: 23773455
references.citation: Biochem J. 2002 Sep 15;366(Pt 3):689-704
references.pmid: 12061891
references.citation: Nucleic Acids Res. 2012 Jul;40(Web Server issue):W537-41
references.pmid: 22570416
references.citation: Proc Natl Acad Sci U S A. 2014 Mar 25;111(12):4560-5
references.pmid: 24616501
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: J Appl Crystallogr. 2009 Apr 1;42(Pt 2):342-346
references.pmid: 27630371
references.citation: Proteins. 2006 Jun 1;63(4):928-38
references.pmid: 16493626
references.citation: Cell Host Microbe. 2009 Jun 18;5(6):571-9
references.pmid: 19527884