title: Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K fromPorphyromonas gingivalis, a causative agent of gum disease from the human oral microbiome.
abstract: Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such asPorphyromonas gingivalis, the main etiological agent of periodontitis.P. gingivalissecretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-β protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys129) likely occupying the S1specificity pocket and exerting latency. Lys129mutation to glutamate or arginine led to misfolded protein that was degradedin vivoMutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129within the S1pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen,i.e.both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain intrans.Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.
authors.affiliation: From the Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, United Kingdom.
authors.forename: Anja
authors.initials: A
authors.lastname: Pomowski
authors.affiliation: the Proteolysis Lab and Crystallographic Methods Lab, Structural Biology Unit, "María de Maeztu" Unit of Excellence, Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas, 08028 Barcelona, Catalonia, Spain.|the Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Catalonia, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Usón
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland, and.
authors.forename: Zuzanna
authors.initials: Z
authors.lastname: Nowakowska
authors.affiliation: the Department of Oral Immunology and Infectious Disease, University of Louisville School of Dentistry, Louisville, Kentucky 40202.
authors.forename: Florian
authors.initials: F
authors.lastname: Veillard
authors.affiliation: the Department of Oral Immunology and Infectious Disease, University of Louisville School of Dentistry, Louisville, Kentucky 40202.
authors.forename: Maryta N
authors.initials: MN
authors.lastname: Sztukowska
authors.affiliation: the Proteolysis Lab and Crystallographic Methods Lab, Structural Biology Unit, "María de Maeztu" Unit of Excellence, Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas, 08028 Barcelona, Catalonia, Spain.
authors.forename: Tibisay
authors.initials: T
authors.lastname: Guevara
authors.affiliation: the Proteolysis Lab and Crystallographic Methods Lab, Structural Biology Unit, "María de Maeztu" Unit of Excellence, Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas, 08028 Barcelona, Catalonia, Spain.
authors.forename: Theodoros
authors.initials: T
authors.lastname: Goulas
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland, and.
authors.forename: Danuta
authors.initials: D
authors.lastname: Mizgalska
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland, and.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Nowak
authors.affiliation: the Department of Oral Immunology and Infectious Disease, University of Louisville School of Dentistry, Louisville, Kentucky 40202.
authors.forename: Barbara
authors.initials: B
authors.lastname: Potempa
authors.affiliation: From the Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, United Kingdom.
authors.forename: James A
authors.initials: JA
authors.lastname: Huntington
authors.affiliation: the Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland, and jan.potempa@louisville.edu.|the Department of Oral Immunology and Infectious Disease, University of Louisville School of Dentistry, Louisville, Kentucky 40202.
authors.forename: Jan
authors.initials: J
authors.lastname: Potempa
authors.affiliation: the Proteolysis Lab and Crystallographic Methods Lab, Structural Biology Unit, "María de Maeztu" Unit of Excellence, Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas, 08028 Barcelona, Catalonia, Spain, fxgr@ibmb.csic.es.
authors.forename: F Xavier
authors.initials: FX
authors.lastname: Gomis-Rüth
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016866:Bacteroidaceae Infections / Q000201:enzymology / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000080867:Gingipain Cysteine Endopeptidases; D005891:Gingivitis / Q000201:enzymology / Q000235:genetics; D006801:Humans; D064307:Microbiota; D009055:Mouth / Q000382:microbiology; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D000072417:Protein Domains; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D037521:Virulence Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D004792:Enzyme Precursors; D000080867:Gingipain Cysteine Endopeptidases; D037521:Virulence Factors; D003546:Cysteine Endopeptidases
keywords: X-ray crystal structure; cysteine protease; host-microbiome interaction; oral microbiota; protein crystallization; protein degradation; protein domain; protein structure; proteinase; zymogenic latency