Contribution of peptide bonds to inhibitor-protease binding: Crystal structures of the turkey ovomucoid third domain backbone variants OMTYK3-Pro18l and OMTKY3-psi[COO]-Leu18l in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY3-psi[CH2NH2+]-Asp19l

X-ray crystallography has been used to determine the 3D structures of two c omplexes between Streptomyces griseus proteinase B (SGPB), a bacterial seri ne proteinase, and backbone variants of turkey ovomucoid third domain (OMTK Y3). The natural P-1 residue (Leu18I) has been substituted by a proline res idue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Th r17I and Leu18I was replaced by an ester bond (OMTKY3-Psi [COO]-Leu18I). Bo th variants lack the P-1 NH group that donates a bifurcated hydrogen bond t o the carbonyl O of Ser214 and O-gamma of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibito rs. The SGPB:OMTKY3-Pro18I complex has many structural differences in the v icinity of the S-1 pocket when compared with the previously determined stru cture of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. Ln contrast, very little structural difference e xists between the complexes of SGPB:OMTKY3-Psi [ECOO]-Leu18I and SGPB:OMTKY 3-Leu18I, aside from an ester O replacing the P-1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P-1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 hav ing a reduced peptide bond between P-1 Leu18I and P'(1) Asp19I, (OMTKY3-psi [CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibi tor. Therefore, its poor ability to inhibit serine proteinases is likely du e to the disruptions of the canonical interactions at the oxyanion hole. (C ) 2001 Academic Press.