A structure-derived snap-trap mechanism of a multispecific serpin from the dysbiotic human oral microbiome

Abstract

Enduring host-microbiome relationships are based on adaptive strategies within a particular ecological niche. Tannerella forsythia is a dysbiotic member of the human oral microbiome that inhabits periodontal pockets and contributes to chronic periodontitis. To counteract endopeptidases from the host or microbial competitors, T. forsythia possesses a serpin-type proteinase inhibitor called miropin. Although serpins from animals, plants, and viruses have been widely studied, those from prokaryotes have received only limited attention. Here we show that miropin uses the serpin-type suicidal mechanism. We found that, similar to a snap trap, the protein transits from a metastable native form to a relaxed triggered or induced form after cleavage of a reactive-site target bond in an exposed reactive-center loop. The prey peptidase becomes covalently attached to the inhibitor, is dragged 75 Å apart, and is irreversibly inhibited. This coincides with a large conformational rearrangement of miropin, which inserts the segment upstream of the cleavage site as an extra β-strand in a central β-sheet. Standard serpins possess a single target bond and inhibit selected endopeptidases of particular specificity and class. In contrast, miropin uniquely blocked many serine and cysteine endopeptidases of disparate architecture and substrate specificity owing to several potential target bonds within the reactive-center loop and to plasticity in accommodating extra β-strands of variable length. Phylogenetic studies revealed a patchy distribution of bacterial serpins incompatible with a vertical descent model. This finding suggests that miropin was acquired from the host through horizontal gene transfer, perhaps facilitated by the long and intimate association of T. forsythia with the human gingiva.

Keywords: inhibition mechanism; inhibitor; molecular biology; peptidase; periodontal disease; protease; protease inhibitor; protein structure; proteinase; proteolysis.

Figure 1.

Miropin complexes with serine or cysteine peptidases. A, SDS-PAGE analysis of complexes. Wild-type or V367K/K368A mutant miropin were incubated for various time points with peptidases in an equimolar ratio or with 2-fold molar excess of the inhibitor. Incubation periods varied between 30 s and 1 h. Complexes with subtilisin, papain, and trypsin with the mutant were further purified by gel filtration. Stars, high molecular mass complexes; black arrows, miropin in complex with peptidase-derived peptides after autolysis; open arrows, unbound peptidases; dotted frames, miropin fragments from the respective RSB to the C terminus. B, peptidase cleavage sites within miropin at the N terminus (panel (i)) and within the RCL of the wild type (panel (ii)) and double-point mutants V367K/K368A (panel (iii)) and K368A/T369K (panel (iv)). Mutated residues are shown in red. Vertical arrows pinpoint the cleavage sites of the distinct peptidases. Try, trypsin; Sub, subtilisin; Pap, papain; HNE, human neutrophil elastase; Elp, porcine pancreatic elastase.

Figure 2.

Effect of reactive-site residue scrambling on miropin inhibition. Inhibition of trypsin was assayed by wild-type inhibitor (A), double mutant V367K/K368A (B), and double mutant K368A/T369K (C). Inhibition was characterized by determining the SI (upper panels) and the association rate constant (k_ass) (lower panels). The data shown are the means and standard deviation of the means from three replicates.

Figure 3.

Structures of native and induced miropin. A, native miropin in the reference front view (according to Ref. 32) as a Richardson-type plot, with strands as arrows and helices as purple spirals (labeled hA–hH and hRCL). The strands are arranged in three β-sheets (sheet sA, yellow strands s6A, s5A, and s3A–s1A; sheet sB, orange strands s0B–s6B; and sheet C, red strands s1C–s4C). The RCL (Glu³⁵³–Pro³⁷⁶) connects strand s5A with s1C and is subdivided into the hinge region (Glu³⁵³–Val³⁶¹; brown ribbon) and the exposed loop (Thr³⁶²–Pro³⁷⁶; blue ribbon). The residues flanking the theoretic RSB (P₁–P₁′; Thr³⁶⁹–Ser³⁷⁰) are shown for their side chains and labeled. The position of the P₁₆ (Glu³⁵³) and P₁₇ (Glu³⁵⁴) residues is indicated by lines. B, same as A but showing trypsin-induced wild-type miropin as representative of the induced miropin structures. β-Strand s4A from sheet sA, absent in A, is shown in the colors of the corresponding segment of A and labeled. Dipeptide Asp₁₉₄–Ser₁₉₅ of trypsin is covalently attached through atom Ser₁₉₅ Oγ to the carbonyl of Lys³⁶⁸ after cleavage of bond P₂–P₁ (Lys³⁶⁸–Thr³⁶⁹). On the primed side, the chain is only defined from Ser³⁷³ (P₄) onwards. C, superposition of native and subtilisin-induced wild-type miropin as Cα-traces in cross-eye stereo depicting sheet sA and the RCL (respectively, in orchid and turquoise), and the flap consisting of helix hF and downstream loop hF-s3A (pink and blue). Upon induction, rotation around the curved orange arrow leads to strand insertion, which entails a left shift for strands s6A and s5A and a right shift for s3A, s2A, and s1A (small orange arrows). The visible ends of the cleaved region of induced miropin (Thr³⁶⁹, P₁ and Pro³⁷⁶, P₇) are pinpointed by black arrows. D and E, topology scheme of native (D) and induced (E) miropin in the coloring of A and B. Relevant positions for the mechanism are depicted in the notation of Schechter and Berger (40) (P₁₈–P₈′; miropin residues Asp³⁵²–Ile³⁷⁷; see also Fig. 7 in Ref. 27). The exposed loop of the RCL (blue coil) spans P₈–P₇′ (Thr³⁶²–Pro³⁷⁶), includes helix hRCL, and contains the theoretic RSB (P₁–P₁′). The hinge region of the RCL (brown coil) spans P₁₇–P₉ (Glu³⁵³–Val³⁶¹). Upon productive cleavage at the RCL and induction (red scissors, D), P₁₅–P₂ (Gly³⁵⁵–Lys³⁶⁸; trypsin cleavage at P₂–P₁) or P₁₅–P₁ (Gly³⁵⁵–Thr³⁶⁹; subtilisin cleavage at P₁–P₁′) becomes inserted into sheet A as s4A (in brown/blue) between s3A and s5A. The residues of each regular secondary structural element are indicated in italics, and those differing in native and induced miropin are in red. The covalently attached endopeptidase is symbolized by a green and yellow ellipse. F, fragment of refined native miropin depicting the five strands of sheet A (from left to right, s6A, s5A, s3A, s2A, and s1A), strand s1C in magenta, and the RCL in orange superposed with the final refined (2mF_obs − DF_calc)-type Fourier map (turquoise mesh) shown with a zone radius of 2 Å and a contour level of 1 σ. Residues Glu³⁵³ (fulcrum) and Gly³⁵⁴ (rotation around bond Cα-C leads to strand insertion) are labeled, and the rotation occurring in the latter upon induction is pinpointed by a magenta arrow. G, same as F but showing trypsin-induced miropin around sheet A only (from left to right, strands s6A–s1A) as magenta sticks, except for strand s4A and the preceding hinge region, in orange as in F. Trypsin dipeptide Asp₁₉₄–Ser₁₉₅ is shown as blue sticks at the sheet bottom. H, same as G but showing the structure of subtilisin-induced miropin. I, same as G but depicting the structure of trypsin-induced miropin V367K/K368A.

Figure 4.

Effect of disulfide bond Cys²⁴⁵–Cys²⁴⁶ on protein stability. A, detail in cross-eye stereo of the structure of trypsin-induced miropin centered on the disulfide bond Cys²⁴⁵–Cys²⁴⁶ superimposed with the final (2mF_obs − DF_calc)-type Fourier map. B, WT and C245A/C246A mutant miropin were subjected to differential scanning calorimetry. The thermograms of temperature versus molar heat capacity (MHC) are representative of triplicate experiments.

Figure 5.

Phylogenetic studies. A, multiple alignment of fragments of selected serpin sequences including T. forsythia miropin (GenBank^TM code WP_041590947). The amino acid numbering and the secondary structure elements—yellow arrows for β-strands and green rods for α-helices—correspond to miropin. Red squares identify the 51 conserved residues in serpins (67). Sequence stretches with low similarity are replaced by three black dots over a salmon background. Gram-negative bacteria (in parentheses, the UniProt codes, except for those preceded by GB, GenBank^TM code): Bacteroides ovatus (A7LR73), Bacteroides uniformis (R9HWW7), Bacteroides vulgatus (I8ZJ35), Arthrospira platensis (D4ZTF4), Anabaena variabilis (Q3M416), Porphyromonas cangingivalis (A0A0A2EV67), Porphyromonas crevioricanis (A0A0A2FPU3), Porphyromonas uenonis (GB WP_025883851), Rhizobium leguminosarium (I9N4F0), Tannerella sp. oral taxon_1 (W2C1Q2), Tannerella sp. oral taxon_2 (W2CNM8) and Chondromyces crocatus (A0A0K1ECH9); Gram-positive bacteria: Legionella feeleii (A0A0W0TMA7), Mycobacterium ulcerans (X8FN27), Streptomyces albus (M9SZH9), Bifidobacterium bifidum (E1AWB1), Bacillus subtilis (A0A0K6L0C3), and Clostridium butyricum (A0A0S3L3I1); eukaryotes: Drosophila erecta (B3NAL5), Homo sapiens_1 (α₁-proteinase inhibitor; P01009), H. sapiens_2 (SCCA-1; P29508), H. sapiens_3 (neuroserpin; Q99574), Rattus norvegicus (D3ZJK2), and Zonotrichia albicollis (GB XP_005481738). B, circular phylogenetic tree reflecting evolutionary distances among the sequences shown in A. Potential bacterial proteins from the Fibrobacteres, Chlorobi, and Bacteroides group are depicted with a blue and yellow background. Other bacterial and eukaryotic sequences are shown over gray and red backgrounds, respectively. The bar represents 0.5 substitutions per site.