Theodoros Goulas

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.

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.

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/β-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.

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

Significance Proteolytic enzymes are inhibited in vivo by protein inhibitors. Such inhibitors are used by symbiotic bacteria in our gut to protect themselves from digestive peptidases. This is the case for Escherichia coli, which has acquired a large, multidomain inhibitor of broad inhibitory spectrum [Escherichia coli α2-macroglobulin (ECAM)]. We studied ECAM and found it is cleaved by host peptidases, which triggers large conformational rearrangement of the inhibitor—shown by protein crystallography and electron microscopy reconstructions—as well as covalent binding of the peptidase. The latter is blocked similarly to a mouse by a snap trap, which prevents damage to the bacterial envelope. Prey peptidases, however, can still be active in the digestion of intake proteins. 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 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 β-propeller domains and a C-terminal β-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.

The pCri System: A Vector Collection for Recombinant Protein Expression and Purification

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5′end and multiple cloning sites at the 3′end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

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.

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 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.

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

Abstract 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.

Structure of RagB, a major immunodominant outer-membrane surface receptor antigen of Porphyromonas gingivalis.

Porphyromonas gingivalis is the main causative agent of periodontitis. It deregulates the inflammatory and innate host immune responses through virulence factors, which include the immunodominant outer-membrane surface receptor antigens A (PgRagA) and B (PgRagB), co-transcribed from the rag pathogenicity island. The former is predicted to be a Ton-dependent porin-type translocator but the targets of this translocation and the molecular function of PgRagB are unknown. Phenomenologically, PgRagB has been linked with epithelial cell invasion and virulence according to murine models. It also acts as a Toll-like receptor agonist and promotes multiple mediators of inflammation. Hence, PgRagB is a candidate for the development of a periodontitis vaccine, which would be facilitated by the knowledge of its atomic structure. Here, we crystallized and solved the structure of 54-kDa PgRagB, which revealed a single domain centered on a curved helical scaffold. It consists of four tetratrico peptide repeats (TPR1-4), each arranged as two helices connected by a linker, plus two extra downstream capping helices. The concave surface bears four large intertwined irregular inserts (A-D), which contribute to an overall compact moiety. Overall, PgRagB shows substantial structural similarity with Bacteroides thetaiotaomicron SusD and Tannerella forsythia NanU, which are, respectively, engaged in binding and uptake of malto-oligosaccharide/starch and sialic acid. This suggests a similar sugar-binding function for PgRagB for uptake by the cognate PgRagA translocator, and, consistently, three potential monosaccharide-binding sites were tentatively assigned on the molecular surface.