Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

Abstract

The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known1,2. Here we determine binding sites, bound conformations and specific drug–receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation–π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, ‘orthosteric’ ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator’s allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

This is a preview of subscription content

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: Structurally diverse allosteric modulators bind spontaneously to the human M2 muscarinic acetylcholine receptor in simulation, revealing a common binding mode.
Figure 2: Experimental validation of computationally derived binding modes.
Figure 3: Mechanisms of positive and negative allosteric modulation.

References

  1. 1

    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  PubMed  PubMed Central  Google Scholar 

  2. 2

    Keov, P., Sexton, P. M. & Christopoulos, A. Allosteric modulation of G protein-coupled receptors: a pharmacological perspective. Neuropharmacology 60, 24–35 (2011)

    CAS  PubMed  Google Scholar 

  3. 3

    Filmore, D. It’s a GPCR world. Modern Drug Discov. 7, 24–28 (2004)

    CAS  Google Scholar 

  4. 4

    Jakubik, J. & El-Fakahany, E. E. Allosteric modulation of muscarinic acetylcholine receptors. Pharmaceuticals 3, 2838–2860 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 6091, 232–236 (2012)

    ADS  Google Scholar 

  7. 7

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Lazareno, S. & Birdsall, N. J. Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors. Mol. Pharmacol. 48, 362–378 (1995)

    CAS  PubMed  Google Scholar 

  9. 9

    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)

    ADS  CAS  PubMed  Google Scholar 

  10. 10

    Shan, Y. et al. How does a drug molecule find its target binding site? J. Am. Chem. Soc. 133, 9181–9183 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Buch, I., Giorgino, T. & De Fabritiis, G. Complete reconstruction of an enzyme-inhibitor binding process by molecular dynamics simulations. Proc. Natl Acad. Sci. USA 108, 10184–10189 (2011)

    ADS  CAS  PubMed  Google Scholar 

  12. 12

    Prilla, S., Schrobang, J., Ellis, J., Höltje, H. D. & Mohr, K. Allosteric interactions with muscarinic acetylcholine receptors: complex role of the conserved tryptophan M2422Trp in a cryptical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity. Mol. Pharmacol. 70, 181–193 (2006)

    CAS  PubMed  Google Scholar 

  13. 13

    Huang, X.-P., Prilla, S., Mohr, K. & Ellis, J. Critical amino acid residues of the common allosteric site on the M2 muscarinic acetylcholine receptor. Mol. Pharmacol. 68, 769–778 (2005)

    CAS  PubMed  Google Scholar 

  14. 14

    May, L. T. et al. Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors. Mol. Pharmacol. 72, 463–476 (2007)

    CAS  PubMed  Google Scholar 

  15. 15

    Trankle, C. et al. Interactions of orthosteric and allosteric ligands with [3H]dimethyl-W84 at the common allosteric site of muscarinic M2 receptors. Mol. Pharmacol. 64, 180–190 (2003)

    PubMed  Google Scholar 

  16. 16

    Ballesteros, J. & 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  Google Scholar 

  17. 17

    Matsui, H., Lazareno, S. & Birdsall, N. J. Probing of the location of the allosteric site on m1 muscarinic receptors by site-directed mutagenesis. Mol. Pharmacol. 47, 88–98 (1995)

    CAS  PubMed  Google Scholar 

  18. 18

    Ma, L. et al. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation. Proc. Natl Acad. Sci. USA 106, 15950–15955 (2009)

    ADS  CAS  PubMed  Google Scholar 

  19. 19

    Daiss, J. O. et al. N+/Si replacement as a tool for probing the pharmacophore of allosteric modulators of muscarinic M2 receptors: synthesis, allosteric potency, and positive cooperativity of silicon-based W84 derivatives. Organometallics 21, 803–811 (2002)

    CAS  Google Scholar 

  20. 20

    Choe, H. W. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011)

    ADS  CAS  PubMed  Google Scholar 

  21. 21

    Bock, A. et al. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling. Nat. Commun. 3, 1044 (2012)

    ADS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Shoichet, B. & Kobilka, B. Structure-based drug screening for G-protein-coupled receptors. Trends Pharmacol. Sci. 33, 268–272 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Totrov, M. & Abagyan, R. Flexible ligand docking to multiple receptor conformations: a practical alternative. Curr. Opin. Struct. Biol. 18, 178–184 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Avlani, V., May, L. T., Sexton, P. M. & Christopoulos, A. Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 308, 1062–1072 (2004)

    CAS  PubMed  Google Scholar 

  25. 25

    Gao, Z.-G. et al. Identification of essential residues involved in the allosteric modulation of the human A3 adenosine receptor. Mol. Pharmacol. 63, 1021–1031 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Silvano, E. et al. The tetrahydroisoquinoline derivative SB269,652 is an allosteric antagonist at dopamine D3 and D2 receptors. Mol. Pharmacol. 78, 925–934 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Lazareno, S., Popham, A. & Birdsall, N. J. Analogs of WIN 62,577 define a second allosteric site on muscarinic receptors. Mol. Pharmacol. 62, 1492–1505 (2002)

    CAS  PubMed  Google Scholar 

  28. 28

    Yanamala, N. & Klein-Seetharaman, J. Allosteric modulation of G protein coupled receptors by cytoplasmic, transmembrane, and extracellular ligands. Pharmaceuticals 3, 3324–3342 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Shaw, D. E. et al. Millisecond-scale molecular dynamics simulation 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?id=1654099 (2009)

  30. 30

    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  PubMed  Google Scholar 

  31. 31

    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)

    ADS  CAS  PubMed  Google Scholar 

  32. 32

    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)

    ADS  CAS  PubMed  Google Scholar 

  33. 33

    Everett, A. J., Openshaw, H. T. & Smith, G. F. The constitution of aspidospermine. Part III. Reactivity at the nitrogen atoms, and biogenetic considerations. J. Chem. Soc. 1120–1123. (1957)

  34. 34

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Shaw, D. E. et al. Atomic-level characterization of the structural dynamics of proteins. Science 330, 341–346 (2010)

    ADS  CAS  PubMed  Google Scholar 

  37. 37

    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)

    Google Scholar 

  38. 38

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

    ADS  CAS  Google Scholar 

  39. 39

    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  PubMed  Google Scholar 

  40. 40

    Bourne, P. E., Ginell, S. L., Low, B. W. & Lessinger, L. Structure of a potent neuromuscular blocking agent: caracurine-II dimethochloride octahydrate, [C40H44N4O2]2+·2Cl·8H2O. J. Cryst. Spectroscop. Res. 15, 453–471 (1985)

    CAS  Google Scholar 

  41. 41

    DeLano, W. L. The PyMOL Molecular Graphics System v. 1.5.0.3-01 (Schrödinger, LLC, New York, New York, 2012)

  42. 42

    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  PubMed  Google Scholar 

  43. 43

    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  PubMed  PubMed Central  Google Scholar 

  44. 44

    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  PubMed  PubMed Central  Google Scholar 

  45. 45

    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 

  46. 46

    Caldwell, J. & Kollman, P. Cation–π interactions: nonadditive effects are critical in their accurate representation. J. Am. Chem. Soc. 117, 4177–4178 (1995)

    CAS  Google Scholar 

  47. 47

    Schneider, H. et al. Host-guest supramolecular chemistry. 34. The incremental approach to noncovalent interactions: Coulomb and van der Waals effects in organic ion pairs. J. Am. Chem. Soc. 20, 7698–7703 (1991)

    Google Scholar 

  48. 48

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

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    ADS  CAS  Google Scholar 

  50. 50

    Gnagey, A. L., Seidenberg, M. & Ellis, J. Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors. Mol. Pharmacol. 56, 1245–1253 (1999)

    CAS  PubMed  Google Scholar 

  51. 51

    Voigtländer, U. et al. Allosteric site on muscarinic acetylcholine receptors: identification of two amino acids in the muscarinic M2 receptor that account entirely for the M2/M5 subtype selectivities of some structurally diverse allosteric ligands in N-methylscopolamine-occupied receptors. Mol. Pharmacol. 64, 21–31 (2003)

    PubMed  Google Scholar 

  52. 52

    Avlani, V. A. et al. Critical role for the second extracellular loop in the binding of both orthosteric and allosteric G protein-coupled receptor ligands. J. Biol. Chem. 282, 25677–25686 (2007)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank T. Mildorf, A. Kruse and B. Kobilka for comments; J. Klepeis, B. Gregersen, J.-L. Li, K. Palmo, A. Donchev and particularly A. Taube for advice and support related to force fields and quantum mechanical calculations; Z. Fan for assistance with statistical analysis; A. Lerer and T. O’Donnell for assistance with simulation and analysis software; A. Philippsen for creating the video; A. Stewart for assistance with mutagenesis and cell line generation; K. Ban and J. Harjani for assistance with chemical synthesis; J. Swarbrick for recording and analysing the two-dimensional NMR data; B. Sleebs and S. Marcuccio for provision of synthetic reagents; J. Dang for advice on analytical chemistry; and M. Kirk and R. Kastleman for editorial assistance. Portions of this work were financed by Program Grant no. 519461 from the National Health and Medical Research Council (NHMRC) of Australia, with synthetic chemistry infrastructure support from the Australian Federal Education Investment Fund Super Science Initiative and Victoria’s Science Agenda Investment Fund. A.C. and P.M.S. are Principal Research Fellows of the NHMRC; J.B.B. is a Senior Research Fellow of the NHMRC; J.R.L. is a Career Development Awardee of the NHMRC.

Author information

Affiliations

Authors

Contributions

R.O.D. conceived this study and, with D.E.S., oversaw molecular dynamics simulations and analysis. R.O.D., H.F.G., D.W.B., J.R.V., A.C.P. and D.H.A. designed and analysed molecular dynamics simulations. H.F.G., J.R.V., A.C.P. and D.H.A. performed molecular dynamics simulations. R.O.D., H.F.G., D.W.B. and J.R.V. performed computational design of receptor mutants and of the modulator 4P-C7/3-phth. C.V. performed all biological assays and, with J.R.V. and A.C., analysed experimental data. M.C. and J.R.L. performed mutagenesis and generated the stable cell lines. J.B.B. and R.R. designed, planned and executed the synthesis of 4P-C7/3-phth, with active input from D.W.B. P.M.S. and A.C. supervised the cell-based biological studies. R.O.D., H.F.G., D.W.B., A.C. and D.E.S. wrote the manuscript. R.O.D., A.C. and D.E.S. supervised the overall research.

Corresponding authors

Correspondence to Ron O. Dror, Arthur Christopoulos or David E. Shaw.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-25, Supplementary Tables 1-8, Supplementary Methods and additional references. (PDF 5767 kb)

The allosteric modulator C7/3-phth binds spontaneously to the M2 receptor in an unbiased molecular dynamics simulation

For clarity, the lipid bilayer, ions, and water molecules are not shown. The video speeds up 44-fold after the modulator binds at 120 ns; the amount of simulated time between successive video frames is 1.08 ns before this point and 47.5 ns afterwards. The Cartesian components of the protein Cα positions were smoothed using Fourier-based Gaussian smoothing (σ = 7.2 ns). Ligand coordinates were not smoothed before the ligand bound, but once it bound, both the Cartesian components of its atom positions and its internal angles were smoothed using Gaussian filters (σ = 7.2 ns). The video was created using OpenStructure17. This is simulation 1 under condition A (Supplementary Table 2). (MP4 2760 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Dror, R., Green, H., Valant, C. et al. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs. Nature 503, 295–299 (2013). https://doi.org/10.1038/nature12595

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

Quick links

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