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.

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.

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 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.

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.

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.

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.

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."

Differential inhibition of restriction enzyme cleavage by chromophore-modified analogues of the antitumour antibiotics mithramycin and chromomycin reveals structure-activity relationships.

Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A(3) (CRO) and six chromophore-modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts. Changes in DNA melting temperature produced by these molecules were also analyzed, as well as their antiproliferative activities against a panel of colon, ovarian and prostate human carcinoma cell lines. Moreover, the cellular uptake of several analogues was examined to identify whether intracellular retention was related to cytotoxicity. These experimental approaches provided mutually consistent evidence of a seeming correlation between the strength of binding to DNA and the antiproliferative activity of the chromophore-modified molecules. Four of the analogues (mithramycin SK, mithramycin SDK, chromomycin SK and chromomycin SDK) showed promising biological profiles.