Abstract
G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.
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Acknowledgements
This work was supported by a joint grant from Pfizer Global Research and Development and from the MRCT Development Gap Fund to C.G.T. and R.H., in addition to core funding from the MRC. G.F.X.S. was financially supported by a Human Frontier Science Project (HFSP) programme grant (RG/0052), a European Commission FP6 specific targeted research project (LSH-2003-1.1.0-1) and an ESRF long-term proposal. J.G.B. is funded by a Wellcome Trust Clinician Scientist Fellowship. We thank E. Ross for his support in the initial stages of the β1AR project at the LMB and for his comments on the manuscript. In addition, we would also like to thank R. Grisshammer, E. Hulme, F. Marshall and M. Weir, as well as J. Li, M. Babu and other colleagues at LMB for their comments. We also thank beamline staff at the European Synchrotron Radiation Facility, particularly C. Riekel and M. Burghammer at ID13 and D. Flot and S. McSweeney at ID 23-2. Finally, we thank D. Loakes for Supplementary Fig. 2.
Author Contributions T.W. devised and carried out receptor expression, purification, crystallization and cryo-cooling of the crystals. Receptor stabilization and baculovirus expression were performed by M.J.S.-V.; both authors were also involved in data collection and preliminary crystallographic analyses of the crystals. P.C.E. helped with the crystal cryo-cooling strategy and in diffraction data collection. J.G.B. performed the functional cAMP and reporter gene assays. R.M. was involved in data collection and processing. A.G.W.L. processed the final data, solved and refined the structure, and assisted with manuscript preparation. The overall project management and manuscript preparation were by R.H., C.G.T. and G.F.X.S.
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G.F.X.S., C.G.T. and R.H. are all on the Scientific Advisory Board of Heptares Therapeutics Ltd.
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The file includes Supplementary Figures 1-10 and Supplementary Tables 1-3. Supplementary Table 1 presents data processing and structure refinement statistics. Supplementary Table 2 presents data on the stability of β1 receptor mutants containing alanine residues throughout cytoplasmic loop 2. Supplementary Table 3 presents the amino acid residues involved in crystal contacts. Supplementary Fig. 1 is an alignment of the primary sequences of turkey β1AR and the human β-adrenergic receptors and Supplementary Fig. 2 shows the structures of agonists and antagonists discussed in the main text. Supplementary Fig. 3 shows G protein coupling data and basal activity for β1AR-m23 in a cell-based assay. Supplementary Figs. 4 and 5 describe the crystallographic packing of receptor molecules and show the location of the ordered detergent molecules. Supplementary Fig. 6 shows the B-factor variation for the structures of rhodopsin, β1AR and β2AR. Supplementary Fig. 7 describes the rmsd according to sequence for the comparison between the β1 and β2 receptors and between molecules A and B of β1AR-m23. Supplementary Fig. 8 depicts electron density maps for the Na+ ion and the ordered water molecule in H6. Supplementary Fig. 9 is an omit map of cytoplasmic loop 2 and Supplementary Fig. 10 shows the extracellular surface charge distribution for the β1 and β2 receptors. (PDF 1484 kb)
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Warne, T., Serrano-Vega, M., Baker, J. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008). https://doi.org/10.1038/nature07101
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DOI: https://doi.org/10.1038/nature07101
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