NMR-STUDIES OF THE MODE OF BINDING OF COREPRESSORS AND INDUCERS TO ESCHERICHIA-COLI TRP REPRESSOR

The binding of the corepressors tryptophan and 5-methyltryptophan and of the inducers 3-indolepropionate, 3-indoleacrylate and 5-methylindol e to the Escherichia coli trp repressor have been studied by H-1-NMR s pectroscopy. Identification of the resonances of the protons of bound ligands and their NOEs to protons of the protein (measured as transfer red NOE) was greatly facilitated by the use of samples of the protein in which the hydrogens of all residues except alanine, isoleucine and threonine were replaced by deuterium. Chemical-shift changes of protei n-backbone resonances and side-chain-amide resonances on ligand bindin g were measured with generally or selectively N-15-labelled protein. T he patterns of changes in the chemical shifts of protein resonances an d, particularly, ligand resonances distinguish the corepressors from t he inducers, indicating, in agreement with earlier work, that corepres sors and inducers bind to the protein in different ways. The NOEs obse rved for the bound ligands have been used to determine the position of the ligands in the crystallographically determined binding site, by m eans of a simulated-annealing molecular-dynamics protocol. The structu res obtained show that the orientation in the binding site of the indo le rings of tryptophan and 5-methyltryptophan and of 3-indolepropionat e and 3-indoleacrylate differ by approximately 180 degrees in solution (in agreement with the crystallographic data for complexes of the trp repressor with tryptophan or with 3-indolepropionate). The value and limitations of calculating ligand positions based on transferred NOE a re discussed.