Structural insights into the subtype-selective antagonist binding to the M₂ muscarinic receptor

Human muscarinic receptor M₂ is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M₂ receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M₂ was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M₂ receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M₂ receptors compared to M₃ receptors leads to subtype selectivity of AF-DX 384.