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

Skewing 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-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 S₁ specificity pocket and exerting latency. Lys¹²⁹ 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 Lys¹²⁹ within the S₁ 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.

Keywords: X-ray crystal structure; cysteine protease; host-microbiome interaction; oral microbiota; protein crystallization; protein degradation; protein domain; protein structure; proteinase; zymogenic latency.

FIGURE 1.

Structure of the Kgp pro-domain. A, ribbon-type plot of Kgp-NPD showing the regular secondary-structure elements (3₁₀-helices in magenta, labeled ηI-ηII; β-strands as blue arrows, labeled βI′ + βI–βIV + βIV′ + βV–βXI; numbering based on the structure of RgpB-NPD; see Fig. 1 in Ref. 43). The inhibitory loop includes residue Lys¹²⁹, which is depicted for its side chain and labeled. B and C, two orthogonal views of A. D, topology scheme of Kgp-NPD roughly in the same orientation as in C. Each regular secondary structure element is labeled and marked with its limiting residues.

FIGURE 2.

Comparison of gingipain pro-domains. A, superposition in cross-eye stereo of Kgp-NPD (blue) and RgpB-NPD (yellow; PDB code 4IEF) (43) in the orientation of Fig. 1C. The respective N and C termini are labeled. B, orthogonal view of A. Here, the proven S₁-intruding residue of RgpB (R₁₂₆) and the putative one of Kgp (K¹²⁹) are shown for their side chains and labeled. C, structure-based sequence alignment of the NPDs of RgpB and Kgp. Residues not defined in the respective structure are in light gray, structurally aligned residues are in black (when differing) or red (when identical), and residues defined but structurally non-equivalent in the two structures are in light salmon. The residue (potentially) intruding the S₁ pocket of the respective CD is framed. Numbering and secondary structure elements (arrows for strands; kringles for helices) above the alignment in green correspond to Kgp; those in blue below the alignment correspond to RgpB.

FIGURE 3.

Effect of Lys¹²⁹ mutation on Kgp expression and activity. Western blotting analysis of parental strain P. gingivalis W83 and mutants K129A, K129E, and K129R is shown. Late exponential/early stationary bacterial cultures (BC) were separated by centrifugation into cell-free growth medium (GM) and cell pellet. The latter was washed, giving rise to the whole cell (WC) fraction, which was further fractionated into the soluble intracellular protein fraction (CP + PP) and the cell envelope fracion (CE) by sonication and ultracentrifugation. A and B, all fractions were standardized to the initial volume of the culture subjected to centrifugation and analyzed by Western blotting to detect Kgp forms (A) and Rgps (control) (B). C and D, Kgp (C) and Rgp (D) gingipain activities determined in whole cultures, cell-free growth medium, and washed cells using specific substrates. The whole-culture activity of the wild-type strain was arbitrarily taken as 100%.

FIGURE 4.

Dimer of Kgp pro-domains. A, two Kgp-NPD moieties, shown as plum and tan ribbons, respectively, associate through lateral attachment of the respective upper sheets of their sandwiches 1 (strands βI, βII, βXI, and βVI; see also Fig. 1D) via a local 2-fold axis (red ellipse) perpendicular to the sheets. The distance between the tips of the two blue arrows is ∼25 Å. B, orthogonal view of A. C, view providing insight into the central part of the hydrophobic core below the upper sheets of sandwiches 1. The side chains of Cys³⁵ of each monomer are depicted and pinpointed by green arrows. The distance between the respective Sγ atoms is ∼7 Å.

FIGURE 5.

Size exclusion chromatography of Kgp-NPD. Recombinant Kgp-NPD (1 mg/ml) was resolved on a Superdex^TM 75 increase 10/300 GL column equilibrated with 50 mm sodium acetate, 150 mm NaCl, pH 5.5 or 6.5, or with 50 mm Tris-HCl, 150 m NaCl, pH 8.0, with each buffer freshly supplemented with 2 mm dithiothreitol. The column was calibrated using conalbumin (75 kDa), ovoalbumin (43 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa), the respective elution volumes are indicated by vertical arrows above the chromatography profiles. For clarity, only the elution profiles at pH 5.5 and 8.0 are shown.

FIGURE 6.

Homology model of zymogenic Kgp. A, ribbon-type plot in cross-eye stereo of the homology model of Kgp-NPD + CD + IgSF in the reference orientation chosen for the RgpB zymogen (see Fig. 2A in Ref. 43). The NPD is in blue, the CD is in hot pink, and the IgSF is in green. The structural calcium and sodium ions from the CD are displayed as red and blue spheres, respectively. Blue and pink arrows pinpoint, respectively, the C-terminal residue of the NPD and the first residue of the CD, which are ∼22 Å apart. The catalytic histidine (His⁴⁴⁴) and the putative inhibitory lysine at the tip of the inhibitory loop (Lys¹²⁹) are displayed for their side chains and labeled, as are the N and C termini of the whole model. B, close-up view of A in mono after a vertical 200° rotation to provide insight into the interaction of the inhibitory loop with the active site cleft of Kgp. The side chain of catalytic cysteine Cys⁴⁷⁷ is further labeled. C, close-up view in mono of B after a horizontal 30° rotation showing the putative inhibitory lysine Lys¹²⁹ penetrating the S₁ pocket of the CD in a similar manner as a l-lysylmethyl moiety (LM) covalently attached to Cys⁴⁷⁷ Sγ (carbon atoms in green) does in the structure of mature Kgp (PDB code 4RBM) (42). D, close-up view of B in cross-eye stereo to highlight the segments putatively rearranged during activation and NPD removal. The parts of the experimental structures of the NPD (cyan) and CD (magenta) deviating significantly are superimposed on the homology model of zymogenic Kgp (encircled 1, Thr⁷⁵–Pro⁸¹; encircled 2, Ser¹²⁶–Lys¹³⁵; encircled 3, Glu¹⁵⁹–Ile¹⁶⁹; encircled 4, Asp²²⁹–Asp²³⁶; encircled 5, Gly⁵²²–Val⁵²⁶; and encircled 6, Ala⁵⁶³–His⁵⁷⁹). In particular, loop Ser¹²⁶–Lys¹³⁵ was adapted so that Lys¹²⁹ matches the l-lysylmethyl moiety shown in C. Green curved arrows highlight the transition from the experimental structures to the homology model.