Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-Ψ[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY3-Ψ[CH 2NH 2+]-Asp19I

Abstract

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P 1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-Ψ[COO]-Leu18I). Both variants lack the P 1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O γ of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S 1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the Δ G° of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-Ψ[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P 1 NH group. Therefore, the difference in Δ G°, 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 having a reduced peptide bond between P 1 Leu18I and P 1′ Asp19I, (OMTKY3-Ψ[CH 2NH 2 +]-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 inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.