Abstract

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.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

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Acknowledgements

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.

Affiliations

  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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Contributions

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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https://doi.org/10.1038/s41589-018-0145-x