Structural insights into binding specificity, efficacy and bias of a β2AR partial agonist

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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Fig. 1: Crystal structure of salmeterol-bound β2AR.
Fig. 2: Structural features of the β2AR–salmeterol–Nb71 complex.
Fig. 3: Salmeterol exosite and receptor-subtype-selectivity determinants.
Fig. 4: Hydrogen-bonding interactions in the orthosteric site and rearrangement of the ligand-binding pocket.
Fig. 5: Ligands and specific residues in the orthosteric site modulate hydrogen-bonding and signaling outcome.
Fig. 6: Spectroscopic interrogation of ligand-induced changes in TM6 conformation on detergent-solubilized, purified labeled receptor.

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.

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.

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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.

Corresponding authors

Correspondence to Cheng Zhang or Brian K. Kobilka.

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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.

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Masureel, M., Zou, Y., Picard, L. et al. Structural insights into binding specificity, efficacy and bias of a β2AR partial agonist. Nat Chem Biol 14, 1059–1066 (2018). https://doi.org/10.1038/s41589-018-0145-x

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