Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases

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Abstract
The Streptomyces subtilisin inhibitor (SSI) is a proteinaceous protease inhibitor which inhibits serine proteases by forming a stable Michaelis complex. The flexible loop region (Thr64–Val69) is a very flexible region in an SSI molecule and its importance in interactions with proteases has been suggested, since conformational change of this loop was found to occur for the smooth binding of SSI with various proteases. In this study, mutated SSIs lacking one or two residues in this region were generated and the effects of deletions on the interaction with proteases were investigated. Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73) both known to be trypsin inhibitors, to examine the effects of deletion on interactions with subtilisin BPN' or trypsin. The deletion of one residue (Gly66) caused increased inhibitory activity toward trypsin, indicating the protruding flexible loop hinders binding with trypsin. Reduction of such hindrance by one-residue shortening in this loop is shown to be effective for the interaction of SSI(Lys73) with trypsin. In contrast, one-residue shortening had virtually no effect on inhibition toward subtilisin BPN'. Differences in the subsite structures of these proteases may have been the reason for this contrast. The deletion of two residues (Thr64 and Gly66) in this region converted SSI into a temporary inhibitor. Structural analysis of the degradation intermediate showed that the peptide bond at the reactive site of doubly deleted SSI was cleaved by subtilisin BPN' after its binding with protease. Thus, the irreversibility of the cleaved peptide bond at the reactive site of mutated SSI in the complex with protease may possibly be the cause for its temporary inhibition. Irregular conformation around the reactive site caused by the deletion of two residues in the flexible loop would convert SSI into a temporary inhibitor. Thus, moderate flexibility in the flexible loop region may possibly be a structural requirement for SSI to function.

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