Abstract
Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.
Highlights
Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria
Molecular dynamics simulation of the complex formation between the catalytic domain of alkaline proteinase (APR) and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR
Bacteria of the genera Pseudomonas, Serratia, and Erwinia secrete homologous 50-kDa proteinases and produce specific, high affinity inhibitors directed against these proteinases that belong to the serralysin branch of the metzincin metalloproteinase superfamily
Summary
Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. Crystal structures of the S. marcescens serralysin-Erwinia chrysanthemi inhibitor Inh and APR1⁄7APRin complex from P. aeruginosa have been determined [9, 10] These structures show that both the Erwinia and the Pseudomonas inhibitors fold into disulfide-stabilized, eight-stranded -barrels with an N-terminal trunk of 10 amino acids with the first five residues of the trunk occupying the extended substrate binding site of the enzyme (see Fig. 1). Ser-1I,2 which is conserved in all known serralysin inhibitors except S. marcescens (where the N terminus is Gly), straddles the S1-S1Ј substrate binding pockets of the enzyme, which allows the ␣-amino and carbonyl groups of the N terminus to chelate the catalytic zinc. The hydroxyl proton forms a hydrogen bond with the hydroxyl oxygen of Tyr-216P and the hydroxyl group of Ser-2I donates a
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