WATER-MOLECULES PARTICIPATE IN PROTEINASE-INHIBITOR INTERACTIONS - CRYSTAL-STRUCTURES OF LEU(18), ALA(18), AND GLY(18) VARIANTS OF TURKEY OVOMUCOID INHIBITOR 3RD-DOMAIN COMPLEXED WITH STREPTOMYCES-GRISEUS PROTEINASE-B

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P-1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala(18), and Gly(18), have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S-1 binding pocket. The number and r elative positions of water molecules bound in the S-1 binding pocket v ary according to the size of the side chain of the P-1 residue. Water molecules in the S-1 binding pocket of SGPB are redistributed in respo nse to the complex formation, probably to optimize hydrogen bonds betw een the enzyme and the inhibitor. There are extensive water-mediated h ydrogen bonds in the interfaces of the complexes. In all complexes, As n 36 of OMTKY3 participates in forming hydrogen bonds, via water molec ules, with residues lining the S-1 binding pocket of SGPB. For a homol ogous series of aliphatic straight side chains, Gly(18), Ala(18), Abu( 18), Ape(18), Ahx(18), and Ahp(18) variants, the binding free energy i s a linear function of the hydrophobic surface area buried in the inte rface of the corresponding complexes. The resulting constant of propor tionality is 34.1 cal mol(-1) A(-2). These structures confirm that the binding of OMTKY3 to the preformed S-1 pocket in SGPB involves no sub stantial structural disturbances that commonly occur in the site-direc ted mutagenesis studies of interior residues in other proteins, thus p roviding one of the most reliable assessments of the contribution of t he hydrophobic effect to protein-complex stability.