The conformational and property space of acetylcholine bound to muscarinic receptors : an entropy component accounts for the subtype selectivity of acetylcholine

The conformational behavior of receptor-bound acetylcholine (ACh) was investigated by molecular dynamics simulations. Based on the great similarity among muscarinic receptors, the study was focused on the human M1, M2, and M5 receptors as previously modeled by us. The results showed that receptor-bound ACh was not frozen in a single preferred conformation but preserved an unexpected fraction of its conformational space. However, there were marked differences between the three receptors since the ligand was mostly trans in the M1 receptor, equally distributed among trans and gauche conformers in M2, and exclusively gauche in the M5; the greater flexibility of M2-bound ACh was paralleled by the greater flexibility of the occupied M2 binding site. By contrast, the property space of receptor-bound ACh, and particularly its virtual (computed, conformation-dependent) lipophilicity, was restricted to relatively narrow ranges optimal for successful interaction. Experimental binding investigations to the individual human M1, M2, and M5 muscarinic receptors showed ACh to have a 10-fold higher affinity for the M2 compared to the M1 and M5 receptors. This selectivity was not confirmed by the calculated binding scores, a fact postulated to be caused by the absence of an entropy component in such binding scores. Indeed, the Shannon entropy of all geometric and physicochemical properties monitored were markedly higher in M2-bound ACh compared to M1-bound and M5-bound ACh. This finding suggests that the selectivity profile of acetylcholine for the M2 receptor is largely entropy-driven, a fact that might explain the intrinsic difficulty to design subtype-selective muscarinic agonists.