Crystal structure at 2.6 Å resolution of the complex of subtilisin BPN′ with Streptomyces subtilisin inhibitor

Abstract

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN′, with its proteinaceous inhibitor SSI ( Streptomyces subtilisin inhibitor) was solved at 2.6 Å resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel β-sheet involving the P 1, P 2 and P 3 residues of the inhibitor, the P 4, P 5 and P 6 residues form an antiparallel β-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN′. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 Å wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the “secondary contact region” of SSI seems to be to support the reactive site loop (“primary contact region”). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle 1978.