Protein Science (2000), 9:83-94. Cambridge Press.

Copyright 2000 The Protein Society

Deleterious effects of $ -branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): Crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB.

Katherine S. Bateman,1 Stephen Anderson,2 Wuyuan Lu,3 M.A.Qasim,4 Michael Laskowski, Jr.,4 and Michael N.G. James1

1Medical Research Council of Canada Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

2Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854

3Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637

4Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

Abstract

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residues becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMYKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three $ -branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 D resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 D resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The $ -branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type ( -branched P1 residue for OMTKY3, Leu18I. i 1 angles of approximately 40o are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60o, -60o, or 180o are more commonly observed but 40o is not unfavorable for the $ -branched side chains. Thr18I O( 1 also forms a hydrogen bond with Ser195 O( in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.