NMR chemical shift mapping of the binding site of a protein proteinase inhibitor: changes in the H-1, C-13 and N-15 NMR chemical shifts of turkey ovomucoid third domain upon binding to bovine chymotrypsin A(alpha)

The substrate-like inhibition of serine proteinases by avian ovomucoid doma ins has provided an excellent model for protein inhibitor-proteinase intera ctions of the standard type, H-1,N-15 and C-13 NMR studies have been undert aken on complexes formed between turkey ovomucoid third domain (OMTKY3)(2) and chymotrypsin A(alpha) (Ctr) in order to characterize structural changes occurring in the Ctr binding site of OMTKY3, N-15 and C-13 were incorporat ed uniformly into OMTKY3, allowing backbone resonances to be assigned for O MTKY3 in both its free and complex states. Chemical shift perturbation mapp ing indicates that the two regions, K13-P22 and N33-A40, are the primary si tes in OMTKY3 involved in Ctr binding, in full agreement with the 12 consen sus proteinase-contact residues of OMTKY3 defined previously on the basis o f X-ray crystallographic and mutational analysis. Smaller chemical shift pe rturbations in selected other regions may result from minor structural chan ges on binding. Through-bond N-15-C-13 correlations between P1-C-13' and P1 '-N-15 in two-dimensional H(N)CO and HN(CO) NMR spectra of selectively labe led OMTKY3 complexed with Ctr indicate that the scissile peptide bond betwe en L18 and E19 of the inhibitor is intact in the complex. The chemical shif ts of the reactive site peptide bond indicate that it is predominantly trig onal, although the data are not inconsistent with a slight perturbation of the hybridization of the peptide bond toward the first tetrahedral state al ong the reaction coordinate. Copyright (C) 2001 John Wiley & Sons, Ltd.