Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist


The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves1,2,3,4,5. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

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Figure 1: The M2 receptor with bound QNB.
Figure 2: Binding interactions between the M2 receptor and QNB.
Figure 3: Convergent evolution of acetylcholine binding sites.
Figure 4: Allosteric binding in the M2 receptor.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for M2-T4L are deposited in the Protein Data Bank under accession code 3UON.


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We thank S. Iwata at Kyoto University for supporting the production of M2 receptor, and we acknowledge support from the Japan Society for the Promotion of Science (Research for Future Program) (T.H.), from the Japan Science and Technology Corporation (CREST) (T.H.), from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid for Scientific Research on Priority Area 15083201 (T.H.), from the Japan Science and Technology Corporation (ERATO) (T.K.), from Toray Science Foundation (T.K.), from Takeda Science Foundation (T.K.), from Ichiro Kanehara Foundation (T.K.), from The Sumitomo Foundation (T.K.), from the National Institutes of Health Grants NS028471 and GM083118 (B.K.K.), from the Mathers Foundation (B.K.K. and W.I.W.), and from the National Science Foundation (A.C.K.). We thank T. S. Kobilka for organizing the GPCR Workshop 2010 that brought together the research groups, and for facilitating this collaboration

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K.H. purified M2 and M2-T4L receptors, characterized their ligand binding activity, and performed attempts to crystallize them with hanging drop and other methods for more than ten years. A.C.K. crystallized the M2-T4L receptors in lipidic cubic phase, collected and processed diffraction data, solved and refined the structure, and assisted with manuscript preparation. H.A. set up the expression system and expressed M2-T4L in large amounts using the insect cell/baculovirus expression system. T.Y.-K. expressed M2 and M2-T4L receptors using a yeast expression system, and purified and crystallized M2 and M2-T4L receptors for five years. M.S. constructed several mutants of M2-T4L and evaluated their stabilities. C.Z. assisted with data collection and processing. W.I.W. oversaw data processing and refinement. T.O. gave advice to K.H. and T.H. on crystallization of the M2 receptor and interpretation of its structure. B.K.K. oversaw lipidic cubic phase crystallization, assisted with data collection, and wrote the manuscript together with T.H. and T.K. T.H., together with K.H., has engaged in biochemical studies of muscarinic receptors for more than thirty years, prepared M2 and M2-T4L receptors, and wrote part of the manuscript. T.K. has been collaborating with T.H. for five years, designed the receptor production strategy with T.H., and wrote part of the manuscript.

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Correspondence to Brian K. Kobilka or Tatsuya Haga or Takuya Kobayashi.

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The authors declare no competing financial interests.

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Haga, K., Kruse, A., Asada, H. et al. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 547–551 (2012).

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