A Comparison of Staphostatin B with Standard Mechanism Serine Protease Inhibitors

Renata Filipek,Matthias Bochtler,Jan Potempa

doi:10.1074/jbc.m411792200

Abstract

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus. We have previously shown that staphostatins A and B are competitive, active site-directed inhibitors that span the active site clefts of their target proteases in the same orientation as substrates. We now report the crystal structure of staphostatin B in complex with wild-type staphopain B at 1.9 A resolution. In the complex structure, the catalytic residues are found in exactly the positions that would be expected for uncomplexed papain-type proteases. There is robust, continuous density for the staphostatin B binding loop and no indication for cleavage of the peptide bond that comes closest to the active site cysteine of staphopain B. The carbonyl carbon atom C of this peptide bond is 4.1 A away from the active site cysteine sulfur Sgamma atom. The carbonyl oxygen atom O of this peptide bond points away from the putative oxyanion hole and lies almost on a line from the Sgamma atom to the C atom. The arrangement is strikingly similar to the "ionmolecule" arrangement for the complex of papain-type enzymes with their substrates but differs significantly from the arrangement conventionally assumed for the Michaelis complex of papain-type enzymes with their substrates and also from the arrangement that is crystallographically observed for complexes of standard mechanism inhibitors and their target serine proteases.

Highlights

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus
Expression, Purification, Crystallization, and Structure Determination—Staphopain B with intact active site and staphostatin B from S. aureus were recombinantly expressed in E. coli and purified as a tight complex
The structure was solved by molecular replacement with MOLREP [25], using the previously solved complex of staphostatin B with an inactive staphopain B mutant, where the active site cysteine was replaced with an alanine (Protein Data Bank accession code 1PXV) as the search model, and was refined with REFMAC [28] (Table I)

Summary

Staphostatin B Versus Standard Mechanism Inhibitors

Bond is about 90° [11], similar to the 105° angle that Burgi and Dunitz found to be associated with short contacts between a nucleophile and a target carbonyl in small molecule crystal structures [12, 13]. Nucleophilic attack could be efficient, but retention of the cleavage products could result in efficient competition between the newly generated amino group and solvent, favoring religation of the inhibitor over cleavage (18 –20). 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]. If not all, inhibitor molecules in the crystal are uncleaved and not covalently linked to protease, and we report an intriguing similarity of the staphopain B-staphostatin B complex with the so-called ion-molecule complex that Kollman and co-workers [22] have suggested for the Michaelis complex of papain-type proteases and their substrates. Our work suggests previously unrecognized similarities and differences between staphostatins and standard mechanism serine protease inhibitors

EXPERIMENTAL PROCEDURES

TABLE I Data collection and refinement statistics

RESULTS

DISCUSSION