Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human β2 adrenoceptor (β2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å/3.7 Å resolution. The cytoplasmic ends of the β2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the β2AR are not seen. The β2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the β2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.
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This study was supported by the Lundbeck Foundation (S.G.F.R.), a National Institutes of Health Ruth L. Kirchstein NRSA grant (D.M.R.), a National Institute of General Medical Sciences grant (W.I.W.), a National Institute of Neurological Disorders and Stroke grant, the Mather Charitable Foundation, and a generous gift from Lundbeck (to B.K.K). G.F.X.S. was financially supported by a Human Frontier Science Project (HFSP) programme grant, a European Commission FP6 specific targeted research project and an ESRF long-term proposal. We thank R. Mackinnon and J. Bowie for advice, R. Stevens for help with early screening efforts, and J. Smith for arranging access to GM/CA-CAT at the APS. Use of the APS is supported by the US Department of Energy. GM/CA-CAT is funded by the US National Institutes of Cancer and General Medical Sciences. We thank X. Deupi and S. Granier for help with data collection. We thank D. Flot for his support at the ID 23.2 microfocus beamline at the European Synchrotron Radiation Facility.
Author Contributions S.G.F.R. performed final stages of β2AR purification, purified Mab5 and prepared Fab5. D.M.R. generated recombinant β2AR used for crystallography. Crystal screening and optimization were performed by S.G.F.R. and D.M.R. H.J.C. assisted with data collection at the Advanced Photon Source, processed all diffraction data and solved the structure of the β2AR–Fab5 complex. F.S.T. expressed β2AR in insect cells and, together with T.S.K., performed the initial stage of β2AR purification. T.S.K. prepared antibody 5. W.I.W. supervised and assisted with data collection at the Advanced Photon Source, and with data processing and structure determination. G.F.X.S. introduced B.K.K. to microfocus diffraction technology and supervised data collection at the European Synchrotron Radiation Facility. P.C.E. and M.B. assisted with data collection at the European Synchrotron Radiation Facility. R.S. and R.F.F. assisted with data collection at the Advanced Photon Source. V.R.P.R. performed the functional characterization of carazolol. B.K.K .was responsible for the overall project management and strategy, and assisted with β2AR purification, crystal harvesting and synchrotron data collection. B.K.K., W.I.W. and G.F.X.S. prepared the manuscript. All authors discussed the results and commented on the manuscript.
The file contains Supplementary Methods, Supplementary Table S1, Supplementary Figures S1-S5 and Legends. Supplementary Table S1 includes X-ray data collection and refinement statistics. Supplementary Figures S1-S3 and S5 show electron density maps for different regions of the crystal structure. Supplementary Figure S4 provides GTPγS binding data demonstrating that carazolol is a partial inverse agonist. This file was modified on 4 November 2007 to correct typographical errors.
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Nature Chemical Biology (2018)