Mutational analysis and molecular modeling of the allosteric binding site of a novel, selective, noncompetitive antagonist of the metabotropic glutamate 1 receptor

Abstract

A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1, 6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F8016.51A, Y8056.55A, and T8157.39M caused complete loss of the [3H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K+ channel current and [Ca2+]i response. The mutation W7986.48F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC50 value (4 versus 128 nM) compared with wild type. The V7575.47L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC50 value (1160 versus 128 nM). Two mutations, N74745.51A and N75045.54A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca2+]i response. We observed a striking conservation in the position of critical residues. The residues Val-7575.47, Trp-7986.48, Phe-8016.51, Tyr8056.55, and Thr-8157.39 are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-7986.48, Phe-8016.51, and Tyr-8056.55, thereby blocking the movement of the TM6 helix, which is crucial for receptor activation.