Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.We acknowledge support from National Institutes of Health grantsNS028471(B.K.K.) andGM56169(W.I.W.), fromtheMathers Foundation (B.K.K.and W.I.W.), and from the National Science Foundation (A.C.K.). This work was supported in part by the Intramural Research Program, NIDDK, NIH, US Department of Health and Human Services. We thank R. Grisshammer and S. Costanzi for advice and discussions during various stages of the project, Y. Zhou for carrying out radioligand binding assays with several M3 receptor–T4 fusion constructs, D. Scarpazza for developing software that enabled forced dissociation simulations, and A. Taube, K. Palmo and D. Borhani for advice related to simulations