Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin.

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo­electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap.

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.

Multiple Architectures and Mechanisms of Latency in Metallopeptidase Zymogens.

Metallopeptidases cleave polypeptides bound in the active-site cleft of catalytic domains through a general base/acid mechanism. This involves a solvent molecule bound to a catalytic zinc and general regulation of the mechanism through zymogen-based latency. Sixty reported structures from 11 metallopeptidase families reveal that prosegments, mostly N-terminal of the catalytic domain, block the cleft regardless of their size. Prosegments may be peptides (5-14 residues), which are only structured within the zymogens, or large moieties (<227 residues) of one or two folded domains. While some prosegments globally shield the catalytic domain through a few contacts, others specifically run across the cleft in the same or opposite direction as a substrate, making numerous interactions. Some prosegments block the zinc by replacing the solvent with particular side chains, while others use terminal α-amino or carboxylate groups. Overall, metallopeptidase zymogens employ disparate mechanisms that diverge even within families, which supports that latency is less conserved than catalysis.

Matrix metalloproteinases outside vertebrates.

The matrix metalloproteinase (MMP) family belongs to the metzincin clan of zinc-dependent metallopeptidases. Due to their enormous implications in physiology and disease, MMPs have mainly been studied in vertebrates. They are engaged in extracellular protein processing and degradation, and present extensive paralogy, with 23 forms in humans. One characteristic of MMPs is a ~165-residue catalytic domain (CD), which has been structurally studied for 14 MMPs from human, mouse, rat, pig and the oral-microbiome bacterium Tannerella forsythia. These studies revealed close overall coincidence and characteristic structural features, which distinguish MMPs from other metzincins and give rise to a sequence pattern for their identification. Here, we reviewed the literature available on MMPs outside vertebrates and performed database searches for potential MMP CDs in invertebrates, plants, fungi, viruses, protists, archaea and bacteria. These and previous results revealed that MMPs are widely present in several copies in Eumetazoa and higher plants (Tracheophyta), but have just token presence in eukaryotic algae. A few dozen sequences were found in Ascomycota (within fungi) and in double-stranded DNA viruses infecting invertebrates (within viruses). In contrast, a few hundred sequences were found in archaea and >1000 in bacteria, with several copies for some species. Most of the archaeal and bacterial phyla containing potential MMPs are present in human oral and gut microbiomes. Overall, MMP-like sequences are present across all kingdoms of life, but their asymmetric distribution contradicts the vertical descent model from a eubacterial or archaeal ancestor. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

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

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 ß-strand in a central ß-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 ß-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.

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.

LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification.

To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion-dominated spectra. This improved protein C terminal-peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or dimethylated lysine and arginine, facilitating detection of these epigenetic modifications.

α2-Macroglobulins: Structure and Function.

α2-macroglobulins are broad-spectrum endopeptidase inhibitors, which have to date been characterised from metazoans (vertebrates and invertebrates) and Gram-negative bacteria. Their structural and biochemical properties reveal two related modes of action: the "Venus flytrap" and the "snap-trap" mechanisms. In both cases, peptidases trigger a massive conformational rearrangement of α2-macroglobulin after cutting in a highly flexible bait region, which results in their entrapment. In some homologs, a second action takes place that involves a highly reactive ß-cysteinyl-γ-glutamyl thioester bond, which covalently binds cleaving peptidases and thus contributes to the further stabilization of the enzyme:inhibitor complex. Trapped peptidases are still active, but have restricted access to their substrates due to steric hindrance. In this way, the human α2-macroglobulin homolog regulates proteolysis in complex biological processes, such as nutrition, signalling, and tissue remodelling, but also defends the host organism against attacks by external toxins and other virulence factors during infection and envenomation. In parallel, it participates in several other biological functions by modifying the activity of cytokines and regulating hormones, growth factors, lipid factors and other proteins, which has a great impact on physiology. Likewise, bacterial α2-macroglobulins may participate in defence by protecting cell wall components from attacking peptidases, or in host-pathogen interactions through recognition of host peptidases and/or antimicrobial peptides. α2-macroglobulins are more widespread than initially thought and exert multifunctional roles in both eukaryotes and prokaryotes, therefore, their on-going study is essential.

Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition.

The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼ 180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric "snap trap."

Structural and functional insight into pan-endopeptidase inhibition by α2-macroglobulins.

Peptidases must be exquisitely regulated to prevent erroneous cleavage and one control is provided by protein inhibitors. These are usually specific for particular peptidases or families and sterically block the active-site cleft of target enzymes using lock-and-key mechanisms. In contrast, members of the +1400-residue multi-domain α2-macroglobulin inhibitor family (α2Ms) are directed against a broad spectrum of endopeptidases of disparate specificities and catalytic types, and they inhibit their targets without disturbing their active sites. This is achieved by irreversible trap mechanisms resulting from large conformational rearrangement upon cleavage in a promiscuous bait region through the prey endopeptidase. After decades of research, high-resolution structural details of these mechanisms have begun to emerge for tetrameric and monomeric α2Ms, which use 'Venus-flytrap' and 'snap-trap' mechanisms, respectively. In the former, represented by archetypal human α2M, inhibition is exerted through physical entrapment in a large cage, in which preys are still active against small substrates and inhibitors that can enter the cage through several apertures. In the latter, represented by a bacterial α2M from Escherichia coli, covalent linkage and steric hindrance of the prey inhibit activity, but only against very large substrates.

Combining phase information in reciprocal space for molecular replacement with partial models.

ARCIMBOLDO allows ab initio phasing of macromolecular structures below atomic resolution by exploiting the location of small model fragments combined with density modification in a multisolution frame. The model fragments can be either secondary-structure elements predicted from the sequence or tertiary-structure fragments. The latter can be derived from libraries of typical local folds or from related structures, such as a low-homology model that is unsuccessful in molecular replacement. In all ARCIMBOLDO applications, fragments are searched for sequentially. Correct partial solutions obtained after each fragment-search stage but lacking the necessary phasing power can, if combined, succeed. Here, an analysis is presented of the clustering of partial solutions in reciprocal space and of its application to a set of different cases. In practice, the task of combining model fragments from an ARCIMBOLDO run requires their referral to a common origin and is complicated by the presence of correct and incorrect solutions as well as by their not being independent. The F-weighted mean phase difference has been used as a figure of merit. Clustering perfect, non-overlapping fragments dismembered from test structures in polar and nonpolar space groups shows that density modification before determining the relative origin shift enhances its discrimination. In the case of nonpolar space groups, clustering of ARCIMBOLDO solutions from secondary-structure models is feasible. The use of partially overlapping search fragments provides a more favourable circumstance and was assessed on a test case. Applying the devised strategy, a previously unknown structure was solved from clustered correct partial solutions.

Expression and purification of integral membrane metallopeptidase HtpX.

Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-ß-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members.

Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog.

Enterotoxigenic Bacteroides fragilis is the most frequent disease-causing anaerobe in the intestinal tract of humans and livestock and its specific virulence factor is fragilysin, also known as B. fragilis toxin. This is a 21-kDa zinc-dependent metallopeptidase existing in three closely related isoforms that hydrolyze E-cadherin and contribute to secretory diarrhea, and possibly to inflammatory bowel disease and colorectal cancer. Here we studied the function and zymogenic structure of fragilysin-3 and found that its activity is repressed by a ∼170-residue prodomain, which is the largest hitherto structurally characterized for a metallopeptidase. This prodomain plays a role in both the latency and folding stability of the catalytic domain and it has no significant sequence similarity to any known protein. The prodomain adopts a novel fold and inhibits the protease domain via an aspartate-switch mechanism. The catalytic fragilysin-3 moiety is active against several protein substrates and its structure reveals a new family prototype within the metzincin clan of metallopeptidases. It shows high structural similarity despite negligible sequence identity to adamalysins/ADAMs, which have only been described in eukaryotes. Because no similar protein has been found outside enterotoxigenic B. fragilis, our findings support that fragilysins derived from a mammalian adamalysin/ADAM xenolog that was co-opted by B. fragilis through a rare case of horizontal gene transfer from a eukaryotic cell to a bacterial cell. Subsequently, this co-opted peptidase was provided with a unique chaperone and latency maintainer in the time course of evolution to render a robust and dedicated toxin to compromise the intestinal epithelium of mammalian hosts.

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.

Frozen fresh blood plasma preserves the functionality of native human α2-macroglobulin.

Human α2-macroglobulin (hα2M) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hα2M undergoes stepwise transitions from its native, expanded, highly flexible, active conformation to an induced, compact, triggered conformation. As a consequence, the peptidase is entrapped by an irreversible Venus flytrap mechanism. Given the importance of hα2M, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hα2M purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hα2M is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hα2M. We therefore used a combination of native and sodium dodecylsulfate polyacrylamide gel electrophoresis, affinity and ion-exchange chromatography, multi-angle laser light scattering after size-exclusion chromatography, free cysteine quantification, and peptidase inhibition assays with endopeptidases of two catalytic classes and three protein substrates, to characterize the biochemical and biophysical properties of hα2M purified ad hoc either from fresh plasma or frozen fresh plasma after thawing. We found no differences in the molecular or functional properties of the preparations, indicating that protective components in plasma maintain native hα2M in a functionally competent state despite freezing.

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.

The crystal structure of human α2-macroglobulin reveals a unique molecular cage.

I'm your Venus: the crystal structure of the human methylamine-induced form of α(2)-macroglobulin (α(2)M) shows its large central cavity can accommodate two medium-sized proteinases. Twelve major entrances provide access for small substrates to the cavity and the still-active trapped "prey". The structure unveils the molecular basis of the unique "venus flytrap" mechanism of α(2)M.

An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution.

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a 'fuzzy complex' that follows a 'dynamic zipper' mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.

Analysis of the inhibiting activity of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) on matrix metalloproteinases.

Matrix metalloproteinases (MMPs) occur in 23 human paralogues with key functions in physiology, and their activity is controlled by protein inhibitors. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is essential for embryogenesis and tumour suppression, has been reported to inhibit MMPs. Here, we developed eukaryotic and bacterial expression systems for different RECK variants and analysed their inhibitory capacity against representative MMPs in vitro. We could not detect any significant inhibition. Instead, we found that partially purified RECK from the conditioned medium of transfected Expi293F cells but not that of ExpiCHO-S or Drosophila Schneider cells contained a contaminant with proteolytic activity. The contaminant was removed through treatment with a small-molecule serine peptidase inhibitor and additional chromatographic purification. A tantamount contaminant was further detected in an equivalent expression system of the N-terminal fragment of the proteoglycan testican 3, but not in those of two other proteins. These results indicate that previous reports of inhibitory activity of recombinant RECK on MMPs, which were performed with partially purified samples, were probably masked by a coeluting contaminant present in the supernatant of HEK293-derived cells. Thus, RECK is probably not a direct inhibitor of MMP catalytic activity but may still regulate MMPs through other mechanisms.