Role of conserved tyrosine lid residues in the activation of the M 2 muscarinic acetylcholine receptor.

The development of subtype-selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues Y104 3.33 , Y403 6.51 , and Y426 7.39 (Ballesteros and Weinstein numbers in superscript) at the human M 2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ~100-fold for the M 2 mAChR, whilst the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M 2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared to the wild-type M 2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand ACh was affected at all mutants containing Y426 7.39 F. These findings will provide insights to consider when designing future mAChR ligands. Significance Statement Prior structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M 2 mAChR had different hydrogen bonding networks in the inactive and active conformations. The role of the hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that the hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.