Abstract
The orexin (also known as hypocretin) G protein–coupled receptors (GPCRs) regulate sleep and other behavioral functions in mammals, and are therapeutic targets for sleep and wake disorders. The human receptors hOX1R and hOX2R, which are 64% identical in sequence, have overlapping but distinct physiological functions and potential therapeutic profiles. We determined structures of hOX1R bound to the OX1R-selective antagonist SB-674042 and the dual antagonist suvorexant at 2.8-Å and 2.75-Å resolution, respectively, and used molecular modeling to illuminate mechanisms of antagonist subtype selectivity between hOX1R and hOX2R. The hOX1R structures also reveal a conserved amphipathic α-helix, in the extracellular N-terminal region, that interacts with orexin-A and is essential for high-potency neuropeptide activation at both receptors. The orexin-receptor crystal structures are valuable tools for the design and development of selective orexin-receptor antagonists and agonists.
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Acknowledgements
We acknowledge support from the Welch Foundation (I-1770 to D.M.R.), the Searle Scholars Program (D.M.R.) and a Packard Foundation Fellowship (D.M.R.). The National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility at the Advanced Photon Source (GM/CA@APS) is funded in whole or in part with Federal funds from the US National Institutes of Health, National Cancer Institute (ACB-12002) and National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. We thank B. Rothermel, L. Lum and L. Zhang (University of Texas Southwestern Medical Center) for materials and assistance with the NFAT-luciferase assay, and S. Lee (University of Texas Southwestern Medical Center) for help with immunofluorescence.
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J.Y. expressed, purified and crystallized the hOX1R-PGS fusion protein, collected diffraction data, solved the structures, carried out ITC experiments and performed NFAT-luciferase assays. K.B. carried out computational docking and simulation experiments for orexin-receptor antagonists. C.A.B. and T.H.S. supervised, designed and analyzed ITC experiments. L.C. and Z.S. assisted with expression and purification of the hOX1R-PGS fusion protein. C.M.H. performed and analyzed radioligand binding assays to measure orexin receptor–ligand affinities. A.J.R. designed and synthesized 2-SORA-DMP. A.L.G., C.J.W., J.J.R. and P.J.C. analyzed docking and simulation experiments for orexin-receptor antagonists and devised experiments to measure receptor function. D.M.R. supervised the overall project, assisted with collection of diffraction data and wrote the manuscript. All authors discussed the results and commented on the manuscript.
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K.B., C.M.H., A.L.G., A.J.R., C.J.W., J.J.R. and P.J.C. are employees of Merck & Co., receive salary and research support from the company and may own stock and/or stock options in the company.
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Supplementary Figure 1 Purification and crystallization of hOX1R-PGS.
(a) Superdex 200 size exclusion profiles of suvorexant-bound hOX1R-PGS (black) and SB-674042-bound hOX1R-PGS (purple).
(b) Coomassie stained PAGE gel of the isolated peak fractions from size exclusion chromatography. Left of markers: SB-674042-bound hOX1R-PGS; Right of markers: suvorexant-bound hOX1R-PGS.
(c) Lipidic cubic phase (LCP) crystallization setup for suvorexant-bound hOX1R-PGS
(d) Equivalent LCP setup for SB-674042-bound hOX1R-PGS.
Supplementary Figure 2 Electron density maps for hOX1R structures.
(a) Stereo view of 2Fo-Fc map, contoured at 1.5 σ, for the region of suvorexant-bound hOX1R containing the ligand, TM2, and TM5.
(b) Stereo view of 2Fo-Fc map, contoured at 1.5 σ, for the equivalent region of the SB-674042-bound hOX1R structure.
Supplementary Figure 3 Electron density and contact maps for the OXR binding pockets.
(a) Stereo view of 2Fo-Fc map, contoured at 1.5 σ, for the residues within 4 Å contact of the ligand in the hOX1R structure bound to SB-674042. Receptor is in blue sticks, ligand is in magenta. At right is a contact map for the antagonist made using LIGPLOT (Wallace, A. et al., Protein Eng. 8, 127-134, 1995).
Average B-factor for contact residues = 41.4 Å2, average B-factor for receptor = 52.8 Å2.
(b) Same as in (a), except for hOX1R (blue) bound to suvorexant (yellow).
Average B-factor for contact residues = 40.9 Å2, average B-factor for receptor = 51.8 Å2.
(c) Same as in (a), except for hOX2R (orange) bound to suvorexant (yellow), pdb 4S0V (Yin, J. et al., Nature 519, 247-250, 2015).
Average B-factor for contact residues = 30.5 Å2, average B-factor for receptor = 41.2 Å2.
Supplementary Figure 4 Molecular dynamics (MD) simulations of suvorexant in hOX1R and hOX2R.
(a) MD simulation trajectory for suvorexant-bound hOX1R. RMSD versus Time is shown for the Cα protein backbone (blue) and the ligand heavy atoms (magenta).
(b) Same as in a, except for suvorexant-bound hOX2R.
Supplementary Figure 5 Binding of 2-SORA-DMP to the orexin receptors.
Radioligand competition assays for 2-SORA-DMP binding to hOX1R (left) or hOX2R (right) membranes. For both panels, n = 3, replicates are from three separate competition assays on the same cell membrane stock. Error bars represent ± s.e.m.
Supplementary Figure 6 Sequence alignment of orexin-receptor N termini.
Alignment includes OX1R and OX2R sequences from Mus musculus (mouse), human, Bos taurus (bovine), Danio rerio (fish), Xenopus laevis (frog), and Canis lupus (dog). The magenta sequences comprise the N-terminal region that is ordered in the hOX1R structures. Sequences were downloaded from the GPCRDB (http://www.gpcr.org/7tm), and alignment was carried out with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2).
Supplementary Figure 7 Cell-surface immunofluorescence staining of orexin-receptor constructs.
Images for transfected CHO-K1 cell surface staining with Alexa488-conjugated M1-anti-FLAG antibody (Sigma) for different orexin receptor constructs (as in Rosenbaum, D.M. et al., Science 318, 1266-1273, 2007). Three separate fields from the same pool of stained cells are shown for each construct.
Supplementary Figure 8 Functional comparison of hOX1R I319 and hOX1R V319.
(a) Dose-response to orexin-A in CHO-K1 cell NFAT-luciferase activation assay. For a and b, n = 3, replicates are from three separate assays on the same transfected cells. Error bars represent ± s.d.
(b) Inhibition of orexin-A (20 nM) signal propagation by increasing concentrations of the antagonist SB-674042.
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Yin, J., Babaoglu, K., Brautigam, C. et al. Structure and ligand-binding mechanism of the human OX1 and OX2 orexin receptors. Nat Struct Mol Biol 23, 293–299 (2016). https://doi.org/10.1038/nsmb.3183
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DOI: https://doi.org/10.1038/nsmb.3183
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