Structure and dynamics of the M3 muscarinic acetylcholine receptor


Acetylcholine, the first neurotransmitter to be identified1, 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 mediate2,3,4. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences5,6. We describe here the structure of the Gq/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 Gi/o-coupled M2 receptor7, 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.

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Figure 1: Major structural features of the M3 receptor.
Figure 2: Orthosteric binding sites of the M2 and M3 receptors.
Figure 3: Molecular dynamics of ligand binding.
Figure 4: G-protein coupling specificity determinants.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for M3–T4L are deposited in the Protein Data Bank (accession code 4DAJ).


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We acknowledge support from National Institutes of Health grants NS028471 (B.K.K.) and GM56169 (W.I.W.), from the Mathers 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.

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A.C.K cloned, expressed, and purified several M3 receptor crystallization constructs; developed the purification procedure; performed crystallization trials, collected diffraction data, solved and refined the structure. J.H. prepared, expressed and characterized various M3 receptor constructs in ligand binding and functional assays. A.C.P. and D.H.A. designed, performed and analysed MD simulations and assisted with manuscript preparation. D.M.R. assisted in design and characterization of initial M3–T4L fusion constructs. E.R. prepared, expressed and tested the pharmacology and stability of several M3 receptor–T4 fusion constructs in insect cells. H.F.G. analysed MD simulations and crystallographic data and assisted with manuscript preparation. T.L. performed binding assays and functional experiments together with J.H. P.S.C. developed and prepared neopentyl glycol detergents used for purifying the M3 receptor. R.O.D. oversaw, designed and analysed MD simulations. D.E.S. oversaw MD simulations and analysis. W.I.W. oversaw refinement of the M3 receptor structure, and assisted in analysis of diffraction data. J.W. provided advice regarding construct design, protein expression and project strategy; and oversaw initial insect cell expression and pharmacological and functional characterization of M3 receptor constructs. B.K.K. was responsible for overall project strategy; guided design of crystallization constructs; and assisted with crystal harvesting and data collection. A.C.K., R.O.D., J.W. and B.K.K. wrote the manuscript.

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Correspondence to Jürgen Wess or Brian K. Kobilka.

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The authors declare no competing financial interests.

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Kruse, A., Hu, J., Pan, A. et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482, 552–556 (2012).

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