Amino-acid sequence and three-dimensional structure of the Staphylococcus aureus metalloproteinase at 1.72 A resolution.

BACKGROUND: Aureolysin is an extracellular zinc-dependent metalloproteinase from the pathogenic bacterium Staphylococcus aureus. This enzyme exhibits in vitro activity against several molecules of biological significance for the host, indicating that it is involved in the pathology of staphylococcal diseases. RESULTS: Here we report the amino-acid sequence and inhibitor-free X-ray crystal structure of aureolysin, a member of the thermolysin family of zinc-dependent metalloproteinases. This enzyme, which binds one zinc and three calcium ions, comprises a single chain of 301 amino acids that consists of a beta-strand-rich upper domain and an alpha-helix-rich lower domain. CONCLUSIONS: The overall structure of aureolysin is very similar to that of the other three members of this family whose structures are known - thermolysin (TLN) from Bacillus thermoproteolyticus, neutral protease (NP) from Bacillus cereus and elastase (PAE) from Pseudomonas aeruginosa. But an important difference has been encountered: in contrast to what has been observed in the other three members of this family (TLN, NP and PAE), inhibitor-free aureolysin displays a 'closed' active site cleft conformation. This new structure therefore raises questions about the universality of the hinge-bending motion model for the neutral metalloproteinases.

Serpin alpha 1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy.

Various conformational forms of the archetypal serpin human alpha 1proteinase inhibitor (alpha 1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because alpha 1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluorescence quenching resolved spectra and red-edge excitation measurements of the Trp environment. Thus, it is possible to define the conformation of the various forms based on the observed fluorescent properties of each of the Trp residues measured under a range of conditions. We show that denaturation in GuHCl, or thermal denaturation in Tris, followed by renaturation, leads to the formation of polymers that contain solvent-exposed Trp 238, which we interpret as ordered head-to-tail polymers (A-sheet polymers). However, thermal denaturation in citrate leads to shorter polymers where some of the Trp 238 residues are not solvent accessible, which we interpret as polymers capped by head-to-head interactions via the C sheet. The latter treatment also generates monomers thought to represent a latent form, but in which the environment of Trp 238 is occluded by ionized groups. These data indicate that the folding pathway of alpha 1PI, and presumably other serpins, is sensitive to solvent composition that affects the affinity of the reactive site loop for the A sheet or the C sheet.

Crystallization and preliminary X-ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H-D-Phe-Phe-Arg-chloromethylketone.

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P2(1)2(1)2(1)) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 A resolution, respectively. Cell dimensions are a = 51.9 A, b = 79.9 A, and c = 99.6 A (P2(1)2(1)2(1)), and a = b = 176.6 A, and c = 143.4 A (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self-rotation function, could accommodate a tetramer in the asymmetric unit.

The role of bacterial and host proteinases in periodontal disease.

It is abundantly obvious that the uncontrolled degradation and/or activation of host defense pathways is the major pathway by which the periodontal pathogen P. gingivalis promotes its growth and proliferation. By being able to shed host receptors, degrade cytokines, and activate coagulation, complement, and kallikrein/kinin pathways it is clear that this organism has found a mechanism(s) to evade host defense and at the same time develop a system for cannibalizing host proteins for its own nutritional usage (Fig 2). Thus, it seems only logical that the development of inhibitors against these bacterial proteinases would be a useful method for negating their activities and making such pathogens more susceptible to attack by host phagocyte cells. In this respect, the structure of the truncated form of RGP has just been elucidated. Thus, it should only be a question of time before inhibitors to this enzyme will be developed and, hopefully, be used to reduce the pathologies associated with the development of periodontitis and/or eliminate the disease altogether.

Role of bacterial proteinases in matrix destruction and modulation of host responses.

Recently accumulated large bodies of evidence have strongly implicated proteolytic enzymes released by subgingival plaque bacteria in the pathogenicity of periodontal disease. With regard to proteolytic power, however, the contribution from different microbial species considered as periodontal pathogens is not equal. Two of these bacteria, P. gingivalis and T. denticola, have developed an elaborate proteolytic systems composed of several surface-located or secreted enzymes, which apparently serve a role to provide bacteria with nutrients in the form of small peptides and amino acids. Of these two species, proteinases of P. gingivalis are the most intensively studied, and during the last decade an impressive array of information has been accumulated with respect to the biochemical characterization of purified proteinases and structure of the genes encoding them, the regulation of expression and the effects of these enzymes on host systems. In addition, studies on proteinase-deficient isogenic mutants has shed light on both their housekeeping functions and potential role(s) in the pathogenicity of periodontitis. Among several proteinases produced by P. gingivalis, the cysteine proteinases, referred to as gingipains, are clearly in the spotlight. They are the subject of several recent reviews and generally considered as the major virulence factors of this periodontal pathogen (59, 105, 139, 182, 183, 186, 281, 284, 286, 289). Gingipains seem to be key players in subverting host defense systems with, significantly, the complement and neutrophils being the main target. In addition, through uncontrolled activation of kallikrein/kinin pathway and coagulation cascade they contribute to local generation of bradykinin and thrombin, two synergistically working pro-inflammatory reagents with a strongly, although indirectly, stimulatory effect on bone resorption. Furthermore, the ability to interact with the cytokine networking systems has the potential to dysregulate the local inflammatory reaction. Finally, gingipains have a strong effect on mechanisms controlling host matrix metalloproteinase activity at the level of gene expression and zymogen activation (Fig. 10). Collectively, at the periodontal lesion site, the non-restrained action of gingipains, supported by other proteinases locally produced by subgingival plaque bacteria, would dysregulate most mechanisms controlling inflammatory reaction. Although successful in limiting infection to the periodontium, the ultimate effect of uncontrolled inflammatory processes would be the destruction of periodontal connective tissue, certainly the hallmark of periodontitis.

Emerging family of proline-specific peptidases of Porphyromonas gingivalis: purification and characterization of serine dipeptidyl peptidase, a structural and functional homologue of mammalian prolyl dipeptidyl peptidase IV.

Porphyromonas gingivalis is an asaccharolytic and anaerobic bacterium that possesses a complex proteolytic system which is essential for its growth and evasion of host defense mechanisms. In this report, we show the purification and characterization of prolyl dipeptidyl peptidase IV (DPPIV) produced by this organism. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. P. gingivalis DPPIV, like its human counterpart, is able to cleave the N terminus of synthetic oligopeptides with sequences analogous to those of interleukins 1beta and 2. Additionally, this protease hydrolyzes biologically active peptides including substance P, fibrin inhibitory peptide, and beta-casomorphin. Southern blot analysis of genomic DNA isolated from several P. gingivalis strains reveal that a single copy of the DPPIV gene was present in all strains tested.

Arginine-specific cysteine proteinase from porphyromonas gingivalis as a convenient tool in protein chemistry.

RgpB, a cysteine proteinase produced by Porphyromonas gingivalis, exhibits proteolytic activity selectively directed against peptide bonds containing an arginine residue in the P1 position. Here we show that this enzyme can be used for very efficient and specific protein cleavage. RgpB is highly active even at high concentrations of denaturing agents, including urea (up to 6 M) and SDS (0.1%), both of them being commonly used for solubilization of insoluble proteins and peptides. Moreover, RgpB is able to digest polypeptide chains in buffers supplemented with 1% Triton X-100, 1% octyl or decylpyranoside, detergents employed for the enzymatic digestion of proteins transferred onto nitrocellulose membranes. These features render RgpB a suitable tool for use in protein chemistry.

Activation of human prothrombin by arginine-specific cysteine proteinases (Gingipains R) from porphyromonas gingivalis.

The effect of 95- (HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated. Each enzyme released thrombin from prothrombin in a dose- and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB. A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that alpha-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments. Significantly, the catalytic efficiency of HRgpA to generate thrombin (k(cat)/K(m) = 1.2 x 10(6) m(-)1 s(-)1) was 100-fold higher than that of RgpB (k(cat)/K(m) = 1.2 x 10(4) m(-)1 s(-)1). The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease. Furthermore, by taking into account that the P. gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B. (1999) Am. Heart J. 138, S534-S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis.

Porphyromonas gingivalis DPP-7 represents a novel type of dipeptidylpeptidase.

A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis. This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of the Staphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, and P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium.

Novel extracellular x-prolyl dipeptidyl-peptidase (DPP) from Streptococcus gordonii FSS2: an emerging subfamily of viridans Streptococcal x-prolyl DPPs.

Streptococcus gordonii is generally considered a benign inhabitant of the oral microflora, and yet it is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Strain FSS2 produced several extracellular aminopeptidase and fibrinogen-degrading activities during growth in culture. In this report we describe the purification, characterization, and cloning of a serine class dipeptidyl-aminopeptidase, an x-prolyl dipeptidyl-peptidase (Sg-xPDPP, for S. gordonii x-prolyl dipeptidyl-peptidase), produced in a pH-controlled batch culture. Purification of this enzyme by anion exchange, gel filtration, and hydrophobic interaction chromatography yielded a protein monomer of approximately 85 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. However, under native conditions, the protein appeared to be a homodimer on the basis of gel filtration and PAGE. Kinetic studies indicated that purified enzyme had a unique and stringent x-prolyl specificity that is comparable to both the dipeptidyl-peptidase IV/CD26 and lactococcal x-prolyl dipeptidyl-peptidase families. Nested PCR cloning from an S. gordonii library enabled the isolation and sequence analysis of the full-length gene. A 759-amino-acid polypeptide with a theoretical molecular mass of 87,115 Da and a calculated pI of 5.6 was encoded by this open reading frame. Significant homology was found with the PepX gene family from Lactobacillus and Lactococcus spp. and putative x-prolyl dipeptidyl-peptidases from other streptococcal species. Sg-xPDPP may serve as a critical factor for the sustained bacterial growth in vivo and furthermore may aid in the proteolysis of host tissue that is commonly observed during SBE pathology.

Isolation and characterization of a highly specific serine endopeptidase from an oral strain of Staphylococcus epidermidis.

Infection by Staphylococcus epidermidis, an opportunistic pathogen, has become a major problem due to the increased use of implanted medical devices and the growing number of patients who are therapeutically or infectiously immunosuppressed. These infections appear to proceed via modulation of the coagulation and complement systems. In this communication we describe the purification and characterization of a novel extracellular proteinase from an oral strain of S. epidermidis that can degrade fibrinogen, complement protein C5, and several other proteins. This proteinase has a strong preference for cleavage after glutamic acid residues, but not after aspartic acid. The S. epidermidis enzyme may be a multifunctional protein which not only provides this organism with both the ability to evade the complement defense system and to dysregulate the coagulation cascade, but also supplies nutrients for its growth through the degradation of Glu-rich proteins.

Rapid and efficient inactivation of IL-6 gingipains, lysine- and arginine-specific proteinases from Porphyromonas gingivalis.

Deregulation of the cytokine network is an important adaptation of pathogenic bacteria to modulate and evade a host immune response. Here we describe that IL-6 is rapidly and efficiently cleaved and inactivated by the arginine- and lysine-specific proteinases from Porphyromonas gingivalis, referred to as RGP-A, RGP-B, and KGP. One of the primary cleavage sites for RGPs has been mapped between R18 and Q19 within the N-terminal region of the IL-6 polypeptide chain; however, both KGP and RGPs cleave IL-6 within the C-terminal region of the polypeptide chain. After these initial proteolytic cleavages, IL-6 is further degraded by each of the enzymes tested. Although KGP is the most potent IL-6-degrading proteinase, the initial C-terminal cleavage of IL-6 mediated by all gingipains is already sufficient to inactivate this cytokine. Our data are consistent with the observation that in periodontitis the IL-6 concentration is lowest in the gingival tissue adjacent to bacterial plaque, whereas significantly elevated concentrations of this cytokine are detected around the infected area. Degradation of IL-6 by gingipains may, therefore, represent an additional mechanism which influences the balance between pro- and anti-inflammatory reactions at distal versus proximal sites from the periodontal plaque.

Prolyl tripeptidyl peptidase from Porphyromonas gingivalis. A novel enzyme with possible pathological implications for the development of periodontitis.

Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. In this report we characterized, both at a protein and genomic level, a novel peptidase of this system with prolyl tripeptidyl peptidase activity. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. The amino acid sequence at the amino terminus and of internal peptide fragments enabled identification of the gene encoding this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl oligopeptidase family of serine proteases. However, it does not share any structural similarity to other tripeptidyl peptidases, which belong to the subtilisin family. The production of prolyl tripeptidyl peptidase may contribute to the pathogenesis of periodontal tissue destruction through the mutual interaction of this enzyme, host and bacterial collagenases, and dipeptidyl peptidases in the degradation of collagen during the course of infection.

Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold.

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain. The catalytic domain is subdivided into two subdomains comprising four- and six-stranded beta-sheets sandwiched by alpha-helices. Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1. The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor.