RELATIVE CONTRIBUTION OF POLAR INTERACTIONS AND CONFORMATIONAL COMPATIBILITY TO THE BINDING OF NEUROKININ-1 RECEPTOR ANTAGONISTS

Based on single residue substitutions, previous studies suggested that Gln165, His197, and His265 of the neurokinin-l receptor interact dire ctly with many nonpeptide antagonists, including CP-96,345. To further test this model, all three residues have been substituted simultaneou sly with alanine. The Q165A-H197A-H265A triple mutant bound CP-96,345 and eight analogs with similar affinity (2-20 mu M), even though the s ame series of compounds bound to the wild-type receptor with affinitie s over a range of 1000-fold. These observations correspond exactly to the prediction of the binding site model. The micromolar binding affin ity of all tested CP-96,345 analogs for the triple mutant seems to ref lect solely van der Waals interactions, which suggests a significant c ontribution of conformational compatibility (or shape complementarity) to binding affinity. The primary role of conformational compatibility in ligand binding was consistent with the observation that simply tra nsferring the residues involved in polar interactions with beta(2)-ago nists into the neurokinin-l receptor did not lead to increased binding affinity for the beta(2)-agonists. Taken together, these results supp ort a general principle of ligand-receptor binding in which specific p olar interactions can take place only if the overall ligand conformati on is compatible with the stereochemistry of the binding pocket. In ad dition, double-residue and triple-residue substitutions, in combinatio n with single-residue substitutions, can provide an alternative route to reveal multiple interactions that may not be detectable by single-r esidue substitutions and represent a novel approach to examine ligand- receptor interactions in the absence of high-resolution structural dat a.