Structure and dynamics of the M3 muscarinic acetylcholine receptor

Abstract

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).

References

  1. 1

    Loewi, O. Über humorale übertragbarkeit der Herznervenwirkung. Pflugers Arch. 189, 239–242 (1921)

    Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Wess, J. Molecular biology of muscarinic acetylcholine receptors. Crit. Rev. Neurobiol. 10, 69–99 (1996)

    CAS  Article  Google Scholar 

  4. 4

    Caulfield, M. P. & Birdsall, N. J. M. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50, 279–290 (1998)

    CAS  PubMed  Google Scholar 

  5. 5

    Conn, P. J., Jones, C. K. & Lindsley, C. W. Subtype-selective allosteric modulators of muscarinic receptors for the treatment of CNS disorders. Trends Pharmacol. Sci. 30, 148–155 (2009)

    CAS  Article  Google Scholar 

  6. 6

    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 

  7. 7

    Haga, K. et al. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature http://dx.doi.org/10.1038/nature10753 (this issue); published online 25 January 2012.

  8. 8

    Yamada, M. et al. Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean. Nature 410, 207–212 (2001)

    CAS  ADS  Article  Google Scholar 

  9. 9

    Poulin, B. et al. The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner. Proc. Natl Acad. Sci. USA 107, 9440–9445 (2010)

    CAS  ADS  Article  Google Scholar 

  10. 10

    Gautam, D. et al. Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth. Proc. Natl Acad. Sci. USA 106, 6398–6403 (2009)

    CAS  ADS  Article  Google Scholar 

  11. 11

    Wess, J., Han, S.-J., Kim, S.-K., Jacobson, K. A. & Li, J. H. Conformational changes involved in G-protein-coupled-receptor activation. Trends Pharmacol. Sci. 29, 616–625 (2008)

    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)

    CAS  ADS  Article  Google Scholar 

  13. 13

    Scarselli, M., Li, B., Kim, S.-K. & Wess, J. Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation. J. Biol. Chem. 282, 7385–7396 (2007)

    CAS  Article  Google Scholar 

  14. 14

    Barnes, P. J. The pharmacological properties of tiotropium. Chest 117, 63S–66S (2000)

    CAS  Article  Google Scholar 

  15. 15

    Casarosa, P., Kiechle, T., Sieger, P., Pieper, M. & Gantner, F. The constitutive activity of the human muscarinic M3 receptor unmasks differences in the pharmacology of anticholinergics. J. Pharmacol. Exp. Ther. 333, 201–209 (2010)

    CAS  Article  Google Scholar 

  16. 16

    Bolden, C., Cusack, B. & Richelson, E. Antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in Chinese hamster ovary cells. J. Pharmacol. Exp. Ther. 260, 576–580 (1992)

    CAS  PubMed  Google Scholar 

  17. 17

    Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein coupled receptors. Methods Neurosci. 25, 366–428 (1995)

    CAS  Article  Google Scholar 

  18. 18

    Li, B. et al. Rapid identification of functionally critical amino acids in a G protein-coupled receptor. Nature Methods 4, 169–174 (2007)

    CAS  Article  Google Scholar 

  19. 19

    Lebon, G., Langmead, C. J., Tehan, B. G. & Hulme, E. C. Mutagenic mapping suggests a novel binding mode for selective agonists of M1 muscarinic acetylcholine receptors. Mol. Pharmacol. 75, 331–341 (2009)

    CAS  Article  Google Scholar 

  20. 20

    Drübbisch, V., Lameh, J., Philip, M., Sharma, Y. K. & Sadée, W. Mapping the ligand binding pocket of the human muscarinic cholinergic receptor Hm1: contribution of tyrosine-82. Pharm. Res. 9, 1644–1647 (1992)

    Article  Google Scholar 

  21. 21

    Dror, R. O. et al. Pathway and mechanism of drug binding to G-protein-coupled receptors. Proc. Natl Acad. Sci. USA 108, 13118–13123 (2011)

    CAS  ADS  Article  Google Scholar 

  22. 22

    Redka, D. S., Pisterzi, L. F. & Wells, J. W. Binding of orthosteric ligands to the allosteric site of the M2 muscarinic cholinergic receptor. Mol. Pharmacol. 74, 834–843 (2008)

    CAS  Article  Google Scholar 

  23. 23

    Valant, C. et al. A novel mechanism of G protein-coupled receptor functional selectivity. J. Biol. Chem. 283, 29312–29321 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Wong, S. K. F. G protein selectivity is regulated by multiple intracellular regions of GPCRs. Neurosignals 12, 1–12 (2003)

    CAS  Article  Google Scholar 

  25. 25

    Blin, N., Yun, J. & Wess, J. Mapping of single amino acid residues required for selective activation of Gq by the M3 muscarinic acetylcholine receptor. J. Biol. Chem. 270, 17741–17748 (1995)

    CAS  Article  Google Scholar 

  26. 26

    Liu, J., Conklin, B. R., Blin, N., Yun, J. & Wess, J. Identification of a receptor/G-protein contact site critical for signaling specificity and G-protein activation. Proc. Natl Acad. Sci. USA 92, 11642–11646 (1995)

    CAS  ADS  Article  Google Scholar 

  27. 27

    Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011)

    CAS  ADS  Article  Google Scholar 

  28. 28

    Blüml, K., Mutschler, E. & Wess, J. Functional role of a cytoplasmic aromatic amino acid in muscarinic receptor-mediated activation of phospholipase C. J. Biol. Chem. 269, 11537–11541 (1994)

    PubMed  Google Scholar 

  29. 29

    MacKerell, A. D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998)

    CAS  Article  Google Scholar 

  30. 30

    Shaw, D. E. et al. Millisecond-scale molecular dynamics simulations on Anton. In Proceedings of the Conference on High Performance Computing, Networking, Storage and Analysis (ACM Press, 2009); available at http://dl.acm.org/citation.cfm?doid=1654059.1654099 (2009)

  31. 31

    Kukkonen, J. P., Näsman, J., Ojala, P., Oker-Blom, C. & Akerman, K. E. Functional properties of muscarinic receptor subtypes Hm1, Hm3 and Hm5 expressed in Sf9 cells using the baculovirus expression system. J. Pharmacol. Exp. Ther. 279, 593–601 (1996)

    CAS  PubMed  Google Scholar 

  32. 32

    Chae, P. S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nature Methods 7, 1003–1008 (2010)

    CAS  Article  Google Scholar 

  33. 33

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

    CAS  Article  Google Scholar 

  34. 34

    Otwinowski, Z. & Minor, W. in Methods Enzymology Vol. 276 (ed. Carter, C. W. Jr ) 307–326 (Academic, 1997)

    Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

  36. 36

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  Article  Google Scholar 

  37. 37

    Davis, I. W., Murray, L. W., Richardson, J. S. & Richardson, D. C. MolProbity: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res. 32, W615–W619 (2004)

    CAS  Article  Google Scholar 

  38. 38

    Schrödinger, L. L. C. The PyMOL Molecular Graphics System, Version 1.3r1 (2010)

    Google Scholar 

  39. 39

    Ward, S. D., Hamdan, F. F., Bloodworth, L. M. & Wess, J. Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy. J. Biol. Chem. 277, 2247–2257 (2002)

    CAS  Article  Google Scholar 

  40. 40

    Bonner, T. I., Buckley, N. J., Young, A. C. & Brann, M. R. Identification of a family of muscarinic acetylcholine receptor genes. Science 237, 527–532 (1987)

    CAS  ADS  Article  Google Scholar 

  41. 41

    Han, S.-J. et al. Pronounced conformational changes following agonist activation of the M3 muscarinic acetylcholine receptor. J. Biol. Chem. 280, 24870–24879 (2005)

    CAS  Article  Google Scholar 

  42. 42

    Dowling, M. R. & Charlton, S. J. Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3 receptor. Br. J. Pharmacol. 148, 927–937 (2006)

    CAS  Article  Google Scholar 

  43. 43

    Ellis, J., Huyler, J. & Brann, M. R. Allosteric regulation of cloned m1-m5 muscarinic receptor subtypes. Biochem. Pharmacol. 42, 1927–1932 (1991)

    CAS  Article  Google Scholar 

  44. 44

    Dror, R. O. et al. Identification of two distinct inactive conformations of the β2-adrenergic receptor reconciles structural and biochemical observations. Proc. Natl Acad. Sci. USA 106, 4689–4694 (2009)

    CAS  ADS  Article  Google Scholar 

  45. 45

    Fahmy, K. et al. Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants. Proc. Natl Acad. Sci. USA 90, 10206–10210 (1993)

    CAS  ADS  Article  Google Scholar 

  46. 46

    Rosenbaum, D. M. et al. Structure and function of an irreversible agonist-β2 adrenoceptor complex. Nature 469, 236–240 (2011)

    CAS  ADS  Article  Google Scholar 

  47. 47

    Kräutler, V., van Gunsteren, W. F. & Hünenberger, P. H. A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J. Comput. Chem. 22, 501–508 (2001)

    Article  Google Scholar 

  48. 48

    Tuckerman, M., Berne, B. J. & Martyna, G. J. Reversible multiple time scale molecular dynamics. J. Chem. Phys. 97, 1990–2001 (1992)

    CAS  ADS  Article  Google Scholar 

  49. 49

    Shan, Y., Klepeis, J. L., Eastwood, M. P., Dror, R. O. & Shaw, D. E. Gaussian split Ewald: A fast Ewald mesh method for molecular simulation. J. Chem. Phys. 122, 54101 (2005)

    ADS  Article  Google Scholar 

  50. 50

    Grubmüller, H., Heymann, B. & Tavan, P. Ligand binding: molecular mechanics calculation of the streptavidin-biotin rupture force. Science 271, 997–999 (1996)

    ADS  Article  Google Scholar 

  51. 51

    Izrailev, S. et al. Molecular dynamics study of unbinding of the avidin-biotin complex. Biophys. J. 72, 1568–1581 (1997)

    CAS  ADS  Article  Google Scholar 

  52. 52

    Mackerell, A. D., Jr, Feig, M. & Brooks, C. L., III Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput. Chem. 25, 1400–1415 (2004)

    CAS  Article  Google Scholar 

  53. 53

    Piana, S., Lindorff-Larsen, K. & Shaw, D. E. How robust are protein folding simulations with respect to force field parameterization? Biophys. J. 100, L47–L49 (2011)

    CAS  Article  Google Scholar 

  54. 54

    Beglov, D. & Roux, B. Finite representation of an infinite bulk system: solvent boundary potential for computer simulations. J. Chem. Phys. 100, 9050–9063 (1994)

    CAS  ADS  Article  Google Scholar 

  55. 55

    Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010)

    CAS  Article  Google Scholar 

  56. 56

    Vanommeslaeghe, K. et al. CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 31, 671–690 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Werner, H.-J. et al. MOLPRO, version 2010.1 (Cardiff University, UK, 2010)

  58. 58

    Tu, T. et al. A scalable parallel framework for analyzing terascale molecular dynamics simulation trajectories. In Proceedings of the 2008 ACM/IEEE Conference on Supercomputing (ACM Press, 2008); available at http://dl.acm.org/citation.cfm?id=1413427 (2008)

  59. 59

    Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)

    CAS  Article  Google Scholar 

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Acknowledgements

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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Contributions

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). https://doi.org/10.1038/nature10867

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