Structural and functional insights into the C-terminal signal domain of the Bacteroidetes type-IX secretion system.

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 ß-sandwich (ß1-ß7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands ß3 and ß4 ('motif Lß3ß4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lß3ß4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl-fluoride inhibitor of serine peptidases.

Peptidases are regulated by latency and inhibitors, as well as compatibilization and cofactors. Ulilysin from Methanosarcina acetivorans, also called lysargiNase, is an archaeal metallopeptidase (MP) that is biosynthesized as a zymogen with a 60-residue N-terminal prosegment (PS). In the presence of calcium, it self-activates to yield the mature enzyme, which specifically cleaves before basic residues and thus complements trypsin in proteomics workflows. Here, we obtained a low-resolution crystal structure of proulilysin, in which 28 protomers arranged as 14 dimers form a continuous double helix of 544 Å pitch that parallels cell axis b of the crystal. The PS includes two α-helices and obstructs the active-site cleft of the catalytic domain (CD) by traversing it in the opposite orientation of a substrate, and a cysteine blocks the catalytic zinc according to a "cysteine-switch mechanism". Moreover, the PS interacts through its first helix with an "S-loop" of the CD, which acts as an "activation segment" that lacks one of two essential calcium cations. Upon PS removal during maturation, the S-loop adopts its competent conformation and binds the second calcium ion. Next, we found that in addition to general MP inhibitors, ulilysin was competitively and reversibly inhibited by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; Ki = 4 µM). This is a compound that normally forms an irreversible covalent complex with serine peptidases but does not inhibit MPs. A high-resolution crystal structure of the complex revealed that the inhibitor penetrates the specificity pocket of ulilysin. A primary amine of the inhibitor salt-bridges an aspartate at the pocket bottom, thus mimicking the basic side chain of substrates. In contrast, the sulfonyl fluoride warhead is not involved and the catalytic zinc ion is freely accessible. Thus, the usage of inhibitor cocktails of peptidases, which typically contain AEBSF at ∼25-fold higher concentrations than the determined Ki, should be avoided when working with ulilysin. Finally, the structure of the complex, which occurred as a crystallographic dimer recurring in previous mature ulilysin structures, unveiled an N-terminal product fragment that delineated the non-primed side of the cleft. These results complement prior structures of ulilysin with primed-side product fragments and inhibitors.

A unique network of attack, defence and competence on the outer membrane of the periodontitis pathogen Tannerella forsythia.

Periodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98-132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct ß-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new "bilobal" or the classic "standard" mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system.

Zymogenic latency in an ∼250-million-year-old astacin metallopeptidase.

The horseshoe crab Limulus polyphemus is one of few extant Limulus species, which date back to ∼250 million years ago under the conservation of a common Bauplan documented by fossil records. It possesses the only proteolytic blood-coagulation and innate immunity system outside vertebrates and is a model organism for the study of the evolution and function of peptidases. The astacins are a family of metallopeptidases that share a central ∼200-residue catalytic domain (CD), which is found in >1000 species across holozoans and, sporadically, bacteria. Here, the zymogen of an astacin from L. polyphemus was crystallized and its structure was solved. A 34-residue, mostly unstructured pro-peptide (PP) traverses, and thus blocks, the active-site cleft of the CD in the opposite direction to a substrate. A central `PP motif' (F35-E-G-D-I39) adopts a loop structure which positions Asp38 to bind the catalytic metal, replacing the solvent molecule required for catalysis in the mature enzyme according to an `aspartate-switch' mechanism. Maturation cleavage of the PP liberates the cleft and causes the rearrangement of an `activation segment'. Moreover, the mature N-terminus is repositioned to penetrate the CD moiety and is anchored to a buried `family-specific' glutamate. Overall, this mechanism of latency is reminiscent of that of the other three astacins with known zymogenic and mature structures, namely crayfish astacin, human meprin ß and bacterial myroilysin, but each shows specific structural characteristics. Remarkably, myroilysin lacks the PP motif and employs a cysteine instead of the aspartate to block the catalytic metal.

De novo design of immunoglobulin-like domains.

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) ß-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture-the non-local cross-ß structure connecting the two ß-sheets-and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.

Repositioning small molecule drugs as allosteric inhibitors of the BFT-3 toxin from enterotoxigenic Bacteroides fragilis.

Bacteroides fragilis is an abundant commensal component of the healthy human colon. However, under dysbiotic conditions, enterotoxigenic B. fragilis (ETBF) may arise and elicit diarrhea, anaerobic bacteremia, inflammatory bowel disease, and colorectal cancer. Most worrisome, ETBF is resistant to many disparate antibiotics. ETBF's only recognized specific virulence factor is a zinc-dependent metallopeptidase (MP) called B. fragilis toxin (BFT) or fragilysin, which damages the intestinal mucosa and triggers disease-related signaling mechanisms. Thus, therapeutic targeting of BFT is expected to limit ETBF pathogenicity and improve the prognosis for patients. We focused on one of the naturally occurring BFT isoforms, BFT-3, and managed to repurpose several approved drugs as BFT-3 inhibitors through a combination of biophysical, biochemical, structural, and cellular techniques. In contrast to canonical MP inhibitors, which target the active site of mature enzymes, these effectors bind to a distal allosteric site in the proBFT-3 zymogen structure, which stabilizes a partially unstructured, zinc-free enzyme conformation by shifting a zinc-dependent disorder-to-order equilibrium. This yields proBTF-3 incompetent for autoactivation, thus ablating hydrolytic activity of the mature toxin. Additionally, a similar destabilizing effect is observed for the activated protease according to biophysical and biochemical data. Our strategy paves a novel way for the development of highly specific inhibitors of ETBF-mediated enteropathogenic conditions.

Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy.

The digestion of gluten generates toxic peptides, among which a highly immunogenic proline-rich 33-mer from wheat α-gliadin, that trigger coeliac disease. Neprosin from the pitcher plant is a reported prolyl endopeptidase. Here, we produce recombinant neprosin and its mutants, and find that full-length neprosin is a zymogen, which is self-activated at gastric pH by the release of an all-ß pro-domain via a pH-switch mechanism featuring a lysine plug. The catalytic domain is an atypical 7+8-stranded ß-sandwich with an extended active-site cleft containing an unprecedented pair of catalytic glutamates. Neprosin efficiently degrades both gliadin and the 33-mer in vitro under gastric conditions and is reversibly inactivated at pH > 5. Moreover, co-administration of gliadin and the neprosin zymogen at the ratio 500:1 reduces the abundance of the 33-mer in the small intestine of mice by up to 90%. Neprosin therefore founds a family of eukaryotic glutamate endopeptidases that fulfils requisites for a therapeutic glutenase.

An engineered protein-based submicromolar competitive inhibitor of the Staphylococcus aureus virulence factor aureolysin.

Aureolysin, a secreted metallopeptidase (MP) from the thermolysin family, functions as a major virulence factor in Staphylococcus aureus. No specific aureolysin inhibitors have yet been described, making this an important target for the development of novel antimicrobial drugs in times of rampant antibiotic resistance. Although small-molecule inhibitors are currently more common in the clinic, therapeutic proteins and peptides (TPs) are favourable due to their high selectivity, which reduces off-target toxicity and allows dosage tuning. The greater wax moth Galleria mellonella produces a unique defensive protein known as the insect metallopeptidase inhibitor (IMPI), which selectively inhibits some thermolysins from pathogenic bacteria. We determined the ability of IMPI to inhibit aureolysin in vitro and used crystal structures to ascertain its mechanism of action. This revealed that IMPI uses the "standard mechanism", which has been poorly characterised for MPs in general. Accordingly, we designed a cohort of 12 single and multiple point mutants, the best of which (I57F) inhibited aureolysin with an estimated inhibition constant (K i) of 346 nM. Given that animals lack thermolysins, our strategy may facilitate the development of safe TPs against staphylococcal infections, including strains resistant to conventional antibiotics.

Intermolecular latency regulates the essential C-terminal signal peptidase and sortase of the Porphyromonas gingivalis type-IX secretion system.

Porphyromonas gingivalis is a keystone pathogen of the human dysbiotic oral microbiome that causes severe periodontitis. It employs a type-IX secretion system (T9SS) to shuttle proteins across the outer membrane (OM) for virulence. Uniquely, T9SS cargoes carry a C-terminal domain (CTD) as a secretion signal, which is cleaved and replaced with anionic lipopolysaccharide by transpeptidation for extracellular anchorage to the OM. Both reactions are carried out by PorU, the only known dual-function, C-terminal signal peptidase and sortase. PorU is itself secreted by the T9SS, but its CTD is not removed; instead, intact PorU combines with PorQ, PorV, and PorZ in the OM-inserted "attachment complex." Herein, we revealed that PorU transits between active monomers and latent dimers and solved the crystal structure of the ∼260-kDa dimer. PorU has an elongated shape ∼130 Å in length and consists of seven domains. The first three form an intertwined N-terminal cluster likely engaged in substrate binding. They are followed by a gingipain-type catalytic domain (CD), two immunoglobulin-like domains (IGL), and the CTD. In the first IGL, a long "latency ß-hairpin" protrudes ∼30 Å from the surface to form an intermolecular ß-barrel with ß-strands from the symmetric CD, which is in a latent conformation. Homology modeling of the competent CD followed by in vivo validation through a cohort of mutant strains revealed that PorU is transported and functions as a monomer through a C690/H657 catalytic dyad. Thus, dimerization is an intermolecular mechanism for PorU regulation to prevent untimely activity until joining the attachment complex.

Structure-based mechanism of cysteine-switch latency and of catalysis by pappalysin-family metallopeptidases.

Tannerella forsythia is an oral dysbiotic periodontopathogen involved in severe human periodontal disease. As part of its virulence factor armamentarium, at the site of colonization it secretes mirolysin, a metallopeptidase of the unicellular pappalysin family, as a zymogen that is proteolytically auto-activated extracellularly at the Ser54-Arg55 bond. Crystal structures of the catalytically impaired promirolysin point mutant E225A at 1.4 and 1.6 Šrevealed that latency is exerted by an N-terminal 34-residue pro-segment that shields the front surface of the 274-residue catalytic domain, thus preventing substrate access. The catalytic domain conforms to the metzincin clan of metallopeptidases and contains a double calcium site, which acts as a calcium switch for activity. The pro-segment traverses the active-site cleft in the opposite direction to the substrate, which precludes its cleavage. It is anchored to the mature enzyme through residue Arg21, which intrudes into the specificity pocket in cleft sub-site S1'. Moreover, residue Cys23 within a conserved cysteine-glycine motif blocks the catalytic zinc ion by a cysteine-switch mechanism, first described for mammalian matrix metallopeptidases. In addition, a 1.5 Šstructure was obtained for a complex of mature mirolysin and a tetradecapeptide, which filled the cleft from sub-site S1' to S6'. A citrate molecule in S1 completed a product-complex mimic that unveiled the mechanism of substrate binding and cleavage by mirolysin, the catalytic domain of which was already preformed in the zymogen. These results, including a preference for cleavage before basic residues, are likely to be valid for other unicellular pappalysins derived from archaea, bacteria, cyanobacteria, algae and fungi, including archetypal ulilysin from Methanosarcina acetivorans. They may further apply, at least in part, to the multi-domain orthologues of higher organisms.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a "CPDCP-trunk" and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the "legumain-binding loop" of CY1 inhibit crayfish astacin following the "raised-elephant-trunk mechanism" recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and ß only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Structure of mammalian plasma fetuin-B and its mechanism of selective metallopeptidase inhibition.

Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallo-peptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked 'CPDCP-trunk', and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWVXGP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel 'raised-elephant-trunk' mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWVXGP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.

Structural determinants of inhibition of Porphyromonas gingivalis gingipain K by KYT-36, a potent, selective, and bioavailable peptidase inhibitor.

Porphyromonas gingivalis is a member of the dysbiotic oral microbiome and a "keystone pathogen" that causes severe periodontal disease, which is among the most prevalent infectious diseases. Part of the virulence factors secreted by P. gingivalis are the essential cysteine peptidases gingipain K (Kgp) and R (RgpA and RgpB), which account for 85% of the extracellular proteolytic activity of the pathogen and are thus prime targets for inhibition. We report the high-resolution (1.20 Å) complex structure of Kgp with KYT-36, a peptide-derived, potent, bioavailable and highly selective inhibitor, which is widely used for studies in vitro, in cells and in vivo. Sub-nanomolar inhibition of Kgp is achieved by tight binding to the active-site cleft, which is covered for its sub-sites S3 through S1' under establishment of nine hydrophobic interactions, 14 hydrogen bonds and one salt bridge. In addition, an inhibitor carbonyl carbon that mimics the scissile carbonyl of substrates is pyramidalized and just 2.02 Å away from the catalytic nucleophile of Kgp, C477Sγ. Thus, the crystal structure emulates a reaction intermediate of the first nucleophilic attack during catalysis of cysteine peptidases. The present study sets the pace for the development of tailored next-generation drugs to tackle P. gingivalis.

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.

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-ß 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.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two ß-propeller domains and a C-terminal ß-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid.

Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S2 and for positively charged residues in S1. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S2 pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.

Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/ß-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a "Michaelis loop" that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants.

A novel mechanism of latency in matrix metalloproteinases.

The matrix metalloproteinases (MMPs) are a family of secreted soluble or membrane-anchored multimodular peptidases regularly found in several paralogous copies in animals and plants, where they have multiple functions. The minimal consensus domain architecture comprises a signal peptide, a 60-90-residue globular prodomain with a conserved sequence motif including a cysteine engaged in "cysteine-switch" or "Velcro" mediated latency, and a catalytic domain. Karilysin, from the human periodontopathogen Tannerella forsythia, is the only bacterial MMP to have been characterized biochemically to date. It shares with eukaryotic forms the catalytic domain but none of the flanking domains. Instead of the consensus MMP prodomain, it features a 14-residue propeptide, the shortest reported for a metallopeptidase, which lacks cysteines. Here we determined the structure of a prokarilysin fragment encompassing the propeptide and the catalytic domain, and found that the former runs across the cleft in the opposite direction to a bound substrate and inhibits the latter through an "aspartate-switch" mechanism. This finding is reminiscent of latency maintenance in the otherwise unrelated astacin and fragilysin metallopeptidase families. In addition, in vivo and biochemical assays showed that the propeptide contributes to protein folding and stability. Our analysis of prokarilysin reveals a novel mechanism of latency and activation in MMPs. Finally, our findings support the view that the karilysin catalytic domain was co-opted by competent bacteria through horizontal gene transfer from a eukaryotic source, and later evolved in a specific bacterial environment.

Structure and mechanism of cysteine peptidase gingipain K (Kgp), a major virulence factor of Porphyromonas gingivalis in periodontitis.

Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys(477)-His(444)-Asp(388), rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates.

Structure-function studies of the staphylococcal methicillin resistance antirepressor MecR2.

Methicillin resistance in Staphylococcus aureus is elicited by the MecI-MecR1-MecA axis encoded by the mec locus. Recently, MecR2 was also identified as a regulator of mec through binding of the methicillin repressor, MecI. Here we show that plasmid-encoded full-length MecR2 restores resistance in a sensitive S. aureus mecR2 deletion mutant of the resistant strain N315. The crystal structure of MecR2 reveals an N-terminal DNA-binding domain, an intermediate scaffold domain, and a C-terminal dimerization domain that contributes to oligomerization. The protein shows structural similarity to ROK (repressors, open reading frames, and kinases) family proteins, which bind DNA and/or sugar molecules. We found that functional cell-based assays of three point mutants affecting residues participating in sugar binding in ROK proteins had no effect on the resistance phenotype. By contrast, MecR2 bound short double-stranded DNA oligonucleotides nonspecifically, and a deletion mutant affecting the N-terminal DNA-binding domain showed a certain effect on activity, thus contributing to resistance less than the wild-type protein. Similarly, a deletion mutant, in which a flexible segment of intermediate scaffold domain had been replaced by four glycines, significantly reduced MecR2 function, thus indicating that this domain may likewise be required for activity. Taken together, these results provide the structural basis for the activity of a methicillin antirepressor, MecR2, which would sequester MecI away from its cognate promoter region and facilitate its degradation.