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

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Hulme, E. C., Birdsall, N. J. & Buckley, N. J. Muscarinic receptor subtypes. Annu. Rev. Pharmacol. Toxicol. 30, 633–673 (1990)

    CAS  Article  Google Scholar 

  2. 2

    Dale, H. H. The action of certain esters and ethers of choline, and their relation to muscarine. J. Pharmacol. Exp. Ther. 6, 147–190 (1914)

    CAS  Google Scholar 

  3. 3

    Haga, K. et al. Functional reconstitution of purified muscarinic receptors and inhibitory guanine nucleotide regulatory protein. Nature 316, 731–733 (1985)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Haga, K. & Haga, T. Purification of the muscarinic acetylcholine receptor from porcine brain. J. Biol. Chem. 260, 7927–7935 (1985)

    CAS  PubMed  Google Scholar 

  5. 5

    Kubo, T. et al. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 323, 411–416 (1986)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Dixon, R. A. et al. Cloning of the gene and cDNA for mammalian β-adrenergic receptor and homology with rhodopsin. Nature 321, 75–79 (1986)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Ovchinnikov, Y. A. Rhodopsin and bacteriorhodopsin: structure–function relationships. FEBS Lett. 148, 179–191 (1982)

    CAS  Article  Google Scholar 

  8. 8

    Wess, J., Eglen, R. M. & Gautam, D. Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development. Nature Rev. Drug Discov. 6, 721–733 (2007)

    CAS  Article  Google Scholar 

  9. 9

    Gregory, K. J., Sexton, P. M. & Christopoulos, A. Allosteric modulation of muscarinic acetylcholine receptors. Curr. Neuropharmacol. 5, 157–167 (2007)

    CAS  Article  Google Scholar 

  10. 10

    Kameyama, K., Haga, K., Haga, T., Moro, O. & Sadée, W. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (β-adrenergic-receptor kinase-1) by a muscarinic receptor M2 mutant lacking phosphorylation sites. Eur. J. Biochem. FEBS 226, 267–276 (1994)

    CAS  Article  Google Scholar 

  11. 11

    Ichiyama, S. et al. The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype. FEBS Lett. 580, 23–26 (2006)

    CAS  Article  Google Scholar 

  12. 12

    Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Jaakola, V. P. et al. The 2.6 Ångstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Chien, E. Y. et al. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330, 1091–1095 (2010)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Shimamura, T. et al. Structure of the human histamine H1 receptor complex with doxepin. Nature 475, 65–70 (2011)

    CAS  Article  Google Scholar 

  17. 17

    Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Hulme, E. C., Lu, Z. L. & Bee, M. S. Scanning mutagenesis studies of the M1 muscarinic acetylcholine receptor. Receptors Channels 9, 215–228 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Heitz, F. et al. Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site. Eur. J. Pharmacol. 380, 183–195 (1999)

    CAS  Article  Google Scholar 

  21. 21

    Wess, J. Mutational analysis of muscarinic acetylcholine receptors: structural basis of ligand/receptor/G protein interactions. Life Sci. 53, 1447–1463 (1993)

    CAS  Article  Google Scholar 

  22. 22

    Goodwin, J. A., Hulme, E. C., Langmead, C. J. & Tehan, B. G. Roof and floor of the muscarinic binding pocket: variations in the binding modes of orthosteric ligands. Mol. Pharmacol. 72, 1484–1496 (2007)

    CAS  Article  Google Scholar 

  23. 23

    Ward, S. D., Curtis, C. A. & Hulme, E. C. Alanine-scanning mutagenesis of transmembrane domain 6 of the M1 muscarinic acetylcholine receptor suggests that Tyr381 plays key roles in receptor function. Mol. Pharmacol. 56, 1031–1041 (1999)

    CAS  Article  Google Scholar 

  24. 24

    Bluml, K., Mutschler, E. & Wess, J. Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors. J. Biol. Chem. 269, 18870–18876 (1994)

    CAS  PubMed  Google Scholar 

  25. 25

    Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Furukawa, H. et al. Conformation of ligands bound to the muscarinic acetylcholine receptor. Mol. Pharmacol. 62, 778–787 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Zacharias, N. & Dougherty, D. A. Cation-π interactions in ligand recognition and catalysis. Trends Pharmacol. Sci. 23, 281–287 (2002)

    CAS  Article  Google Scholar 

  28. 28

    Brams, M. et al. Crystal structures of a cysteine-modified mutant in loop D of acetylcholine-binding protein. J. Biol. Chem. 286, 4420–4428 (2011)

    CAS  Article  Google Scholar 

  29. 29

    Oswald, C. et al. Crystal structures of the choline/acetylcholine substrate-binding protein ChoX from Sinorhizobium meliloti in the liganded and unliganded-closed states. J. Biol. Chem. 283, 32848–32859 (2008)

    CAS  Article  Google Scholar 

  30. 30

    Colletier, J. P. et al. Structural insights into substrate traffic and inhibition in acetylcholinesterase. EMBO J. 25, 2746–2756 (2006)

    CAS  Article  Google Scholar 

  31. 31

    Asada, H. et al. Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis. Microb. Cell Fact. 10, 24 (2011)

    CAS  Article  Google Scholar 

  32. 32

    Weber, W., Weber, E., Geisse, S. & Memmert, K. Optimisation of protein expression and establishment of the Wave Bioreactor for Baculovirus/insect cell culture. Cytotechnology 38, 77–85 (2002)

    CAS  Article  Google Scholar 

  33. 33

    Haga, K. & Haga, T. Affinity chromatography of the muscarinic acetylcholine receptor. J. Biol. Chem. 258, 13575–13579 (1983)

    CAS  PubMed  Google Scholar 

  34. 34

    Haga, T., Haga, K. & Hulme, E. C. in Receptor Biochemistry: A Practical Approach (ed. Hulme, E. C. ) 51–78 (Oxford Univ. Press, 1990)

    Google Scholar 

  35. 35

    Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)

    CAS  Article  Google Scholar 

  36. 36

    Otwinowski, Z. & Minor, W. Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  37. 37

    McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007)

    CAS  Article  Google Scholar 

  38. 38

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  39. 39

    Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. A robust bulk-solvent correction and anisotropic scaling procedure. Acta Crystallogr. D 61, 850–855 (2005)

    Article  Google Scholar 

Download references

Acknowledgements

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

Author information

Affiliations

Authors

Contributions

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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-7 with legends, Supplementary Tables 1-3 and additional references. (PDF 5000 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/nature10753

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing