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NMR chemical shift mapping of the binding site of a protein proteinase inhibitor: changes in the 1 H, 13 C and 15 N NMR chemical shifts of turkey ovomucoid third domain upon binding to bovine chymotrypsin A α
Author(s) -
Song Jikui,
Markley John L.
Publication year - 2001
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.530
Subject(s) - chemical shift , chemistry , scissile bond , stereochemistry , serine proteinase inhibitors , peptide bond , peptide , carbon 13 nmr , crystallography , binding site , active site , nuclear magnetic resonance spectroscopy , solid state nuclear magnetic resonance , enzyme , biochemistry , serine protease , nuclear magnetic resonance , protease , physics
The substrate‐like inhibition of serine proteinases by avian ovomucoid domains has provided an excellent model for protein inhibitor‐proteinase interactions of the standard type. 1 H, 15 N and 13 C NMR studies have been undertaken on complexes formed between turkey ovomucoid third domain (OMTKY3) 2 and chymotrypsin A α (Ctr) in order to characterize structural changes occurring in the Ctr binding site of OMTKY3. 15 N and 13 C were incorporated uniformly into OMTKY3, allowing backbone resonances to be assigned for OMTKY3 in both its free and complex states. Chemical shift perturbation mapping indicates that the two regions, K13‐P22 and N33‐A40, are the primary sites in OMTKY3 involved in Ctr binding, in full agreement with the 12 consensus proteinase‐contact residues of OMTKY3 defined previously on the basis of X‐ray crystallographic and mutational analysis. Smaller chemical shift perturbations in selected other regions may result from minor structural changes on binding. Through‐bond 15 N– 13 C correlations between P1‐ 13 C′ and P1′‐ 15 N in two‐dimensional H(N)CO and HN(CO) NMR spectra of selectively labeled OMTKY3 complexed with Ctr indicate that the scissile peptide bond between L18 and E19 of the inhibitor is intact in the complex. The chemical shifts of the reactive site peptide bond indicate that it is predominantly trigonal, although the data are not inconsistent with a slight perturbation of the hybridization of the peptide bond toward the first tetrahedral state along the reaction coordinate. Copyright © 2001 John Wiley & Sons, Ltd.Abbreviations used : BPTI bovine pancreatic trypsin inhibitor, Swiss‐Prot P00974Ctr bovine chymotrypsin A α Swiss‐Prot P00766OMTKY3 third domain of the proteinase inhibitor (ovomucoid) from turkey ( Meleagris gallopavo ), Swiss‐Prot P01004SSI streptomyces subtilisin inhibitor, Swiss‐Prot P01006STI soybean trypsin inhibitor, Swiss‐Prot P01071.