Contribution of peptide bonds to inhibitor-protease binding: Crystal structures of the Turkey ovomucoid third domain backbone variants OMTKY3-Pro181 and OMTKY3-ψ[COO]-Leu181 in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY3-ψ[CH₂NH₂⁺]-Asp191

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 P₁ 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 P₁ 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 S₁ pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the ΔG° 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 P₁ NH group. Therefore, the difference in ΔG°, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P₁ NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P₁ Leu18I and P′₁ Asp19I, (OMTKY3-Ψ[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 inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.