The Staphostatin-Staphopain Complex

Abstract

Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Our recent crystal structure of staphostatin B has shown that this inhibitor forms a mixed, eight-stranded ␤-barrel with statistically significant similarity to lipocalins, but not to cystatins. We now present the 1.8-Å crystal structure of staphostatin B in complex with an inactive mutant of its target protease. The complex is held together through extensive interactions and buries a total surface area of 2300 Å 2 . Unexpectedly for a cysteine protease inhibitor, staphostatin B binds to staphopain B in an almost substratelike manner. The inhibitor polypeptide chain runs through the protease active site cleft in the forward direction, with residues IG-TS in P2 to P2 positions. Both in the free and complexed forms, the P1 glycine residue of the inhibitor is in a main chain conformation only accessible to glycines. Mutations in this residue lead to a loss of affinity of the inhibitor for protease and convert the inhibitor into a substrate.The emergence of multiple antibiotic resistance in Staphylococci, in particular Staphylococcus aureus, has become a major medical concern. With the prevalence of methicillin-resistant S. aureus in hospitals throughout the world (1, 2) and with the appearance of strains that are resistant to "last resort" glycopeptide antibiotics (3, 4), treatment of S. aureus infections in difficult cases could soon depend on a few new compounds that have either been recently introduced to the clinic or are still in clinical trials (5-9). A possible strategy for the development of new antibacterials is to target bacterial virulence factors. The S. aureus arsenal of virulence factors includes secreted toxins (10, 11), immune modulatory molecules (12), adhesion molecules (13), signaling factors (14, 15), and possibly secreted proteases (16).Staphopains A and B are the major secreted cysteine proteases of S. aureus. The literature contains evidence both for and against their role as virulence factors. Random mutagenesis has shown that transposon insertion into the V8-proteinase gene attenuates S. aureus virulence in three separate animal models (mouse abscess, bacteraemia, and wound infection models) (17). More recent work suggests that loss of virulence could be because of a polar effect of the insertion event on the downstream sspB gene encoding staphopain B (18). Consistent with this, inactivation of the sspB gene attenuates S. aureus virulence in the skin abscess model. In contrast, staphopain A, a close homologue of staphopain B, appears to be dispensable for virulence at least in this model. 1Staphopains A and B are remote members of the papain superfamily of enzymes and are encoded on the genome as preproenzymes in the scp and ssp loci, respectively. Recently, it was shown that a short open reading frame in the ssp operon termed sspC codes for an inhibitor of staphopain B, the cysteine protease in this operon (19). This initial finding could be extended to the scp op...

Regulating Cysteine Protease Activity: Essential Role of Protease Inhibitors as Guardians and Regulators

“…The staphostatin loop residues 97 to 101 bind along the active site cleft in a substrate-like direction completely covering access to the active site of staphopain [197], whereas the exclusion domain reaches into the active site cleft of a papain-like structure with the N-terminus only, filling the S3 and beyond binding sites leaving the rest of the cleft freely accessible to peptidyl substrates.…”

A Comparison of Staphostatin B with Standard Mechanism Serine Protease Inhibitors

“…Our previous crystal structure of staphostatin B in complex with an inactive mutant of staphopain B that had an alanine instead of the active site cysteine established a general similarity between staphostatin B and canonical mechanism serine protease inhibitors (3). As the uncleaved, recombinant inhibitor had been crystallized in complex with inactive enzyme, the binding loop of the inhibitor was obviously uncleaved and not covalently linked to the protease in the crystal structure.…”

“…Staphostatins bind very tightly to their target enzymes; in the case of staphostatin B, the binding to staphopain B is known to be tighter than 5 nM (3). Wild-type staphostatin B is very resistant to cleavage, but mutation of a critical glycine residue in the binding loop converted the inhibitor into a staphopain B substrate that was cleaved immediately downstream of the mutated residue (2,3). We have previously crystallized staphostatin B alone (4) and in complex with an inactive staphopain B mutant (3).…”

“…There are obvious analogies between staphostatins and standard mechanism serine protease inhibitors: (i) they are tight binding, (ii) they interact with their target enzymes in a manner broadly similar to substrates, (iii) their binding loops are exposed and somewhat flexible in the free inhibitor structures (possibly with the exception of bovine pancreatic trypsin inhibitor), (iv) the binding loops rearrange and assume a "canonical" conformation in the presence of the target enzymes, (v) the inhibitors appear largely resistant to cleavage, and (vi) peptides that mimic only the binding loop have much lower affinity than complete inhibitor molecules and behave as substrates, not as inhibitors (3,11).…”

“…Cleaved staphostatins precipitate (21); religation has not been observed to the best of our knowledge. (iv) In contrast to canonical mechanism serine protease inhibitors, staphostatins are highly sensitive to mutations in their binding loops (3). (v) Detection of an acylenzyme intermediate has been reported for the complex of at least one standard mechanism inhibitor in complex with its target protease (11), although it remains to be proven that the acyl-enzyme was formed prior to the denaturation step that was required for analysis.…”