Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K from Porphyromonas gingivalis, a causative agent of gum disease from the human oral microbiome

Abstract

Edited by Joseph JezSkewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis, the main etiological agent of periodontitis. P. gingivalis secretes 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-␤ protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgpregulating 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 (Lys 129 ) likely occupying the S 1 specificity pocket and exerting latency. Lys 129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo. Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys 129 within the S 1 pocket 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 in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.

The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function

“…They are cysteine proteases that hydrolyze peptide bonds at the carboxyl group of arginine (RgpA/B: Arg–Xaa) or lysine residues (Kgp: Lys–Xaa; Pike et al, 1994 ). They are exported into the periplasm as inactive zymogens, with the N-terminal prodomain (NTP) functioning as a chaperone and maintaining the latency of the proteases (Mikolajczyk et al, 2003 ; Pomowski et al, 2017 ). After folding in the periplasm, they are transported to the bacterial surface, where they are subjected to extensive post-translational processing.…”

The Oral Parasitic Microbiome in Hepatitis B Virus Infected Su-danese Patients with Gum Disease

“…E. gingivalis is a protozoan parasite, which belongs to the genus Entamoeba that inhabits the oropharynx and is thought to be a commensally oral microbiome in humans ( 21 – 23 ), it is commonly found in cytological and histopathology specimens, and more common in patients with poor oral hygiene, bad dentition, or with immune suppression. However, the identification of oral parasites microbiome rarely studied in developing countries, especially in the relation of E. gingivalis with infectious diseases such as HBV infection.…”

Pathogenesis of Important Virulence Factors of Porphyromonas gingivalis via Toll-Like Receptors

“…Based on analysis of the high-resolution crystal structure of Kgp competent fragments, the key catalytic mechanism of Kgp probably lies in the requirement of a triplet (C 477 -H 444 -D 388 ) instead of a cysteine-histidine dimer (de Diego et al, 2014; Gorman et al, 2015). In addition, dimerization of the pro-domain also plays a substantial role in the specific latency mechanism of Kgp (Pomowski et al, 2017). In an animal experiment designed to test the efficacy of three gene vaccines ( rgpA, rgpB , and kgp ) in treating peri-implant inflammation, the kgp and rgpA DNA vaccines enhanced immune responses and significantly retarded alveolar bone loss in vivo , whereas the rgpB vaccine was ineffective (Guo et al, 2014).…”