Salmeterol is a partial agonist for the β2 adrenergic receptor (β2AR) and the first long-acting β2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol’s safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β1AR and β2AR explain the high receptor-subtype selectivity. A structural comparison with the β2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited β-arrestin recruitment for salmeterol.

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Data availability

Atomic coordinates and structure factors for the crystal structure have been deposited in the Protein Data Bank under accession code PDB 6CSY. Other data and results are available upon request.

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Change history

  • 30 November 2018

    In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.


  1. 1.

    Kenakin, T. Drug efficacy at G protein–coupled receptors. Annu. Rev. Pharmacol. Toxicol. 42, 349–379 (2002).

  2. 2.

    Zhu, B. T. Rational design of receptor partial agonists and possible mechanisms of receptor partial activation: a theory. J. Theor. Biol. 181, 273–291 (1996).

  3. 3.

    Cazzola, M. & Donner, C. F. Long-acting β2 agonists in the management of stable chronic obstructive pulmonary disease. Drugs 60, 307–320 (2000).

  4. 4.

    Baker, J. G., Proudman, R. G. & Hill, S. J. Salmeterol’s extreme β2 selectivity is due to residues in both extracellular loops and transmembrane domains. Mol. Pharmacol. 87, 103–120 (2015).

  5. 5.

    Ferguson, G. T., Funck-Brentano, C., Fischer, T., Darken, P. & Reisner, C. Cardiovascular safety of salmeterol in COPD. Chest 123, 1817–1824 (2003).

  6. 6.

    Twentyman, O. P., Finnerty, J. P., Harris, A., Palmer, J. & Holgate, S. T. Protection against allergen-induced asthma by salmeterol. Lancet 336, 1338–1342 (1990).

  7. 7.

    Ball, D. I. et al. Salmeterol, a novel, long-acting β2-adrenoceptor agonist: characterization of pharmacological activity in vitro and in vivo. Br. J. Pharmacol. 104, 665–671 (1991).

  8. 8.

    Johnson, M. et al. The pharmacology of salmeterol. Life Sci. 52, 2131–2143 (1993).

  9. 9.

    Calverley, P. M. et al. Salmeterol and fluticasone propionate and survival in chronic obstructive pulmonary disease. N. Engl. J. Med. 356, 775–789 (2007).

  10. 10.

    Wijesinghe, M., Perrin, K., Harwood, M., Weatherall, M. & Beasley, R. The risk of asthma mortality with inhaled long acting β-agonists. Postgrad. Med. J. 84, 467–472 (2008).

  11. 11.

    Weatherall, M., Wijesinghe, M., Perrin, K., Harwood, M. & Beasley, R. Meta-analysis of the risk of mortality with salmeterol and the effect of concomitant inhaled corticosteroid therapy. Thorax 65, 39–43 (2010).

  12. 12.

    Coleman, R. A., Johnson, M., Nials, A. T. & Vardey, C. J. Exosites: their current status, and their relevance to the duration of action of long-acting beta 2-adrenoceptor agonists. Trends Pharmacol. Sci. 17, 324–330 (1996).

  13. 13.

    Clark, R. B., Allal, C., Friedman, J., Johnson, M. & Barber, R. Stable activation and desensitization of beta 2-adrenergic receptor stimulation of adenylyl cyclase by salmeterol: evidence for quasi-irreversible binding to an exosite. Mol. Pharmacol. 49, 182–189 (1996).

  14. 14.

    Green, S. A., Spasoff, A. P., Coleman, R. A., Johnson, M. & Liggett, S. B. Sustained activation of a G protein-coupled receptor via “anchored” agonist binding: molecular localization of the salmeterol exosite within the 2-adrenergic receptor. J. Biol. Chem. 271, 24029–24035 (1996).

  15. 15.

    Isogaya, M. et al. Identification of a key amino acid of the β2-adrenergic receptor for high affinity binding of salmeterol. Mol. Pharmacol. 54, 616–622 (1998).

  16. 16.

    Rong, Y. et al. Probing the salmeterol binding site on the β2-adrenergic receptor using a novel photoaffinity ligand, [125I]iodoazidosalmeterol. Biochemistry 38, 11278–11286 (1999).

  17. 17.

    Gimenez, L. E., Baameur, F., Vayttaden, S. J. & Clark, R. B. Salmeterol efficacy and bias in the activation and kinase-mediated desensitization of β2-adrenergic receptors. Mol. Pharmacol. 87, 954–964 (2015).

  18. 18.

    van der Westhuizen, E. T., Breton, B., Christopoulos, A. & Bouvier, M. Quantification of ligand bias for clinically relevant β2-adrenergic receptor ligands: implications for drug taxonomy. Mol. Pharmacol. 85, 492–509 (2014).

  19. 19.

    Tran, T. M. et al. Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the β2-adrenergic receptor using phosphoserine-specific antibodies. Mol. Pharmacol. 65, 196–206 (2004).

  20. 20.

    Drake, M. T. et al. β-arrestin-biased agonism at the β2-adrenergic receptor. J. Biol. Chem. 283, 5669–5676 (2008).

  21. 21.

    Carter, A. A. & Hill, S. J. Characterization of isoprenaline- and salmeterol-stimulated interactions between β2-adrenoceptors and β-arrestin 2 using β-galactosidase complementation in C2C12 cells. J. Pharmacol. Exp. Ther. 315, 839–848 (2005).

  22. 22.

    Moore, R. H. et al. Salmeterol stimulation dissociates β2-adrenergic receptor phosphorylation and internalization. Am. J. Respir. Cell Mol. Biol. 36, 254–261 (2007).

  23. 23.

    Walker, J. K. & DeFea, K. A. Role for β-arrestin in mediating paradoxical β2AR and PAR2 signaling in asthma. Curr. Opin. Pharmacol. 16, 142–147 (2014).

  24. 24.

    Billington, C. K., Penn, R. B. & Hall, I. P. β2 agonists. Handb. Exp. Pharmacol. 237, 23–40 (2017).

  25. 25.

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

  26. 26.

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

  27. 27.

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

  28. 28.

    Warne, T. et al. The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 469, 241–244 (2011).

  29. 29.

    Ring, A. M. et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013).

  30. 30.

    Nygaard, R. et al. The dynamic process of β2-adrenergic receptor activation. Cell 152, 532–542 (2013).

  31. 31.

    Manglik, A. et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161, 1101–1111 (2015).

  32. 32.

    Sounier, R. et al. Propagation of conformational changes during μ-opioid receptor activation. Nature 524, 375–378 (2015).

  33. 33.

    Gregorio, G. G. et al. Single-molecule analysis of ligand efficacy in β2AR–G-protein activation. Nature 547, 68–73 (2017).

  34. 34.

    Staus, D. P. et al. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol. Pharmacol. 85, 472–481 (2014).

  35. 35.

    Zou, Y., Weis, W. I. Kobilka, B. K. & Seifert, R. N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor. PLoS One 7, e46039 (2012).

  36. 36.

    Caffrey, M. Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu. Rev. Biophys. 38, 29–51 (2009).

  37. 37.

    Baker, J. G. The selectivity of β-adrenoceptor agonists at human β1-, β2- and β3-adrenoceptors. Br. J. Pharmacol. 160, 1048–1061 (2010).

  38. 38.

    Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).

  39. 39.

    Fronik, P., Gaiser, B. I. & Sejer Pedersen, D. Bitopic ligands and metastable binding sites: opportunities for G protein-coupled receptor (GPCR) medicinal chemistry. J. Med. Chem. 60, 4126–4134 (2017).

  40. 40.

    Wieland, K., Zuurmond, H. M., Krasel, C., Ijzerman, A. P. & Lohse, M. J. Involvement of Asn-293 in stereospecific agonist recognition and in activation of the β2-adrenergic receptor. Proc. Natl. Acad. Sci. USA 93, 9276–9281 (1996).

  41. 41.

    Liapakis, G., Chan, W. C., Papadokostaki, M. & Javitch, J. A. Synergistic contributions of the functional groups of epinephrine to its affinity and efficacy at the β2 adrenergic receptor. Mol. Pharmacol. 65, 1181–1190 (2004).

  42. 42.

    Thomsen, A. R. B. et al. GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling. Cell 166, 907–919 (2016).

  43. 43.

    Namkung, Y. et al. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET. Nat. Commun. 7, 12178 (2016).

  44. 44.

    Picard, L.-P., Schönegge, A. M., Lohse, M. J. & Bouvier, M. Bioluminescence resonance energy transfer-based biosensors allow monitoring of ligand- and transducer-mediated GPCR conformational changes. Commun. Biol. 1, 106 (2018).

  45. 45.

    Yao, X. J. et al. The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex. Proc. Natl. Acad. Sci. USA 106, 9501–9506 (2009).

  46. 46.

    Dawaliby, R. et al. Allosteric regulation of G protein–coupled receptor activity by phospholipids. Nat. Chem. Biol. 12, 35–39 (2016).

  47. 47.

    Schafer, C. T., Fay, J. F., Janz, J. M. & Farrens, D. L. Decay of an active GPCR: conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state. Proc. Natl. Acad. Sci. USA 113, 11961–11966 (2016).

  48. 48.

    Fay, J. F. & Farrens, D. L. Purification of functional CB1 and analysis by site-directed fluorescence labeling methods. Methods Enzymol. 593, 343–370 (2017).

  49. 49.

    Doose, S., Neuweiler, H. & Sauer, M. A close look at fluorescence quenching of organic dyes by tryptophan. Chemphyschem 6, 2277–2285 (2005).

  50. 50.

    Cazzola, M., Calzetta, L. & Matera, M. G. β2-adrenoceptor agonists: current and future direction. Br. J. Pharmacol. 163, 4–17 (2011).

  51. 51.

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

  52. 52.

    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).

  53. 53.

    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).

  54. 54.

    Shaw, D. E. et al. Millisecond-scale molecular dynamics simulations on Anton. Proc. Conf. High Perform. Comput. Netw. Storage Anal. (pp. 1–11. ACM, Portland, Oregon, USA, 2009).

  55. 55.

    Shaw, D. E. et al. Anton 2: raising the bar for performance and programmability in a special-purpose molecular dynamics supercomputer. Sc14: Int. Conf. High Perform. Comput., Netw. Storage Anal. (pp. 41–53. IEEE, Hoboken, New Jersey, USA, 2014).

  56. 56.

    Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

  57. 57.

    Choi, U. B. et al. Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex. Nat. Struct. Mol. Biol. 17, 318–324 (2010).

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This work was supported by National Institutes of Health grant R01NS028471 (B.K.K.), Canadian Institute for Health Research foundation grant FDN-148431 (M.B.), an American Heart Association Postdoctoral fellowship (17POST33410958; M.M.) and Predoctoral Fellowship (13PRE17110027; J.P.M.), a studentship from the FRQ-S (L.-P.P.), the NIH Pharmacological Sciences Training Program (T32GM007767; J.P.M.) and the National Institutes of Health MIRA 1R35GM128641-01 (C.Z.). B.K.K. is supported by the Chan Zuckerberg Biohub. M.B. is supported as a Canada Research Chair in Signal Transduction and Molecular Pharmacology. The authors thank J. Gullingsrud for assistance with MD software.

Author information

Author notes

    • Emma van der Westhuizen

    Present address: Monash Institute for Pharmaceutical Sciences, Monash University, Victoria, Australia

    • Jacob P. Mahoney

    Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    • Thomas J. Mildorf

    Present address: Dropbox, New York, NY, USA

    • Ron O. Dror

    Present address: Department of Computer Science and Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, USA

  1. These authors contributed equally: Matthieu Masureel, Yaozhong Zou.


  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    • Matthieu Masureel
    • , Yaozhong Zou
    • , João P. G. L. M. Rodrigues
    • , William I. Weis
    •  & Brian K. Kobilka
  2. Geneus Technologies, Ltd, Chengdu, Sichuan, People’s Republic of China

    • Yaozhong Zou
  3. Department of Biochemistry, Institute for Research in Immunology and Cancer, Université de Montreal, Montreal, Québec, Canada

    • Louis-Philippe Picard
    • , Emma van der Westhuizen
    •  & Michel Bouvier
  4. Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA

    • Jacob P. Mahoney
  5. Department of Computer Science, Stanford University, Stanford, CA, USA

    • João P. G. L. M. Rodrigues
  6. Department of Structural Biology, Stanford University, Stanford, CA, USA

    • João P. G. L. M. Rodrigues
    •  & William I. Weis
  7. D. E. Shaw Research, New York, NY, USA

    • Thomas J. Mildorf
    • , Ron O. Dror
    •  & David E. Shaw
  8. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    • David E. Shaw
  9. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    • Els Pardon
    •  & Jan Steyaert
  10. Structural Biology Research Center, VIB, Brussels, Belgium

    • Els Pardon
    •  & Jan Steyaert
  11. Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, CA, USA

    • Roger K. Sunahara
  12. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    • Cheng Zhang


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C.Z. and Y.Z. expressed and purified the receptor and nanobody for crystallography studies, collected X-ray diffraction data and solved the crystal structure. M.M. developed the Atto655 reporter system; purified and labeled receptors used in fluorescence studies; collected spectroscopic data; and performed radioactive ligand binding assays. L.-P.P. generated mutants of N293 and S204. E.v.d.W. and L.-P.P. performed Gs-activation and β-arrestin2-recruitment BRET assays under supervision from M.B. J.P.M. performed Octet RED experiments under supervision from R.K.S. J.P.G.L.M.R. performed modeling and sampling of the Atto655 dye on the receptor structures. T.J.M. and R.O.D. performed and analyzed MD-simulation studies. R.O.D. and D.E.S. oversaw MD simulations and analysis. E.P. generated the nanobody library and performed the initial selections. J.S. supervised nanobody production. W.I.W. supervised and assisted with the structure refinement. M.M., C.Z. and B.K.K. interpreted data, made figures and wrote the manuscript. B.K.K. provided overall project supervision.

Competing interests

The BRET-based biosensors used in the present study are licensed to Domain Therapeutics but are freely available from M.B. for noncommercial academic use. M.B. is the chair of the Scientific Advisory Board of Domain Therapeutics. B.K.K. is a cofounder of and consultant for ConfometRx.

Corresponding authors

Correspondence to Cheng Zhang or Brian K. Kobilka.

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