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Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant

Abstract

The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans1. Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia2. The human OX2 receptor (OX2R) belongs to the β branch of the rhodopsin family of GPCRs3, and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant4. Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX2R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX2R expands our understanding of peptidergic GPCR ligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.

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Figure 1: Fusion protein engineering and structural features of hOX2R.
Figure 2: Suvorexant interaction with hOX2R.
Figure 3: Docked poses for synthetic orexin receptor antagonists.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structure have been deposited in the PDB under accession 4RNB.

References

  1. Li, J., Hu, Z. & de Lecea, L. The hypocretins/orexins: integrators of multiple physiological functions. Br. J. Pharmacol. 171, 332–350 (2014)

    Article  CAS  PubMed  Google Scholar 

  2. Michelson, D. et al. Safety and efficacy of suvorexant during 1-year treatment of insomnia with subsequent abrupt treatment discontinuation: a phase 3 randomised, double-blind, placebo-controlled trial. Lancet Neurol. 13, 461–471 (2014)

    Article  CAS  PubMed  Google Scholar 

  3. Fredriksson, R., Lagerström, M. C., Lundin, L.-G. & Schiöth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003)

    Article  CAS  PubMed  Google Scholar 

  4. Winrow, C. J. & Renger, J. J. Discovery and development of orexin receptor antagonists as therapeutics for insomnia. Br. J. Pharmacol. 171, 283–293 (2014)

    Article  CAS  PubMed  Google Scholar 

  5. Zhu, Y. et al. Orexin receptor type-1 couples exclusively to pertussis toxin-insensitive G-proteins, while orexin receptor type-2 couples to both pertussis toxin-sensitive and -insensitive G-proteins. J. Pharmacol. Sci. 92, 259–266 (2003)

    Article  CAS  PubMed  Google Scholar 

  6. Lin, L. et al. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98, 365–376 (1999)

    Article  CAS  PubMed  Google Scholar 

  7. Chemelli, R. M. et al. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98, 437–451 (1999)

    Article  CAS  PubMed  Google Scholar 

  8. Nishino, S., Ripley, B., Overeem, S., Lammers, G. J. & Mignot, E. Hypocretin (orexin) deficiency in human narcolepsy. Lancet 355, 39–40 (2000)

    Article  CAS  PubMed  Google Scholar 

  9. Cox, C. D. et al. Discovery of the dual orexin receptor antagonist [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the treatment of insomnia. J. Med. Chem. 53, 5320–5332 (2010)

    Article  CAS  PubMed  Google Scholar 

  10. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into 2-adrenergic receptor function. Science 318, 1266–1273 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Caffrey, M. Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu. Rev. Biophys. 38, 29–51 (2009)

    Article  CAS  PubMed  Google Scholar 

  13. White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Egloff, P. et al. Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc. Natl Acad. Sci. USA 111, E655–E662 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Horcajada, C., Guinovart, J. J., Fita, I. & Ferrer, J. C. Crystal structure of an archaeal glycogen synthase: insights into oligomerization and substrate binding of eukaryotic glycogen synthases. J. Biol. Chem. 281, 2923–2931 (2006)

    Article  CAS  PubMed  Google Scholar 

  16. Manglik, A. et al. Crystal structure of the µ-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Malherbe, P. et al. Mapping the binding pocket of dual antagonist almorexant to human orexin 1 and orexin 2 receptors: comparison with the selective OX1 antagonist SB-674042 and the selective OX2 antagonist N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA). Mol. Pharmacol. 78, 81–93 (2010)

    Article  CAS  PubMed  Google Scholar 

  19. Kruse, A. C. et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482, 552–556 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ballesteros, J. A. Activation of the β2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J. Biol. Chem. 276, 29171–29177 (2001)

    Article  CAS  PubMed  Google Scholar 

  21. Bokoch, M. P. et al. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463, 108–112 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rasmussen, S. G. F. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cox, C. D. et al. Conformational analysis of N,N-disubstituted-1,4-diazepane orexin receptor antagonists and implications for receptor binding. Bioorg. Med. Chem. Lett. 19, 2997–3001 (2009)

    Article  CAS  PubMed  Google Scholar 

  25. Sakurai, T. et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92, 573–585 (1998)

    Article  CAS  PubMed  Google Scholar 

  26. Kolb, P. et al. Structure-based discovery of β2-adrenergic receptor ligands. Proc. Natl Acad. Sci. USA 106, 6843–6848 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Brisbare-Roch, C. et al. Promotion of sleep by targeting the orexin system in rats, dogs and humans. Nature Med. 13, 150–155 (2007)

    Article  CAS  PubMed  Google Scholar 

  28. Malherbe, P. et al. Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX2 receptor. Br. J. Pharmacol. 156, 1326–1341 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Langmead, C. J. et al. Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptor. Br. J. Pharmacol. 141, 340–346 (2004)

    Article  CAS  PubMed  Google Scholar 

  30. Tran, D.-T. et al. Chimeric, mutant orexin receptors show key interactions between orexin receptors, peptides and antagonists. Eur. J. Pharmacol. 667, 120–128 (2011)

    Article  CAS  PubMed  Google Scholar 

  31. Kobilka, B. K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231, 269–271 (1995)

    Article  CAS  PubMed  Google Scholar 

  32. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  PubMed  Google Scholar 

  34. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cowtan, K. Fitting molecular fragments into electron density. Acta Crystallogr. D 64, 83–89 (2008)

    Article  CAS  PubMed  Google Scholar 

  37. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Painter, J. & Merritt, E. A. TLSMD web server for the generation of multi-group TLS models. J. Appl. Crystallogr. 29, 109-–111 (2006)

    Google Scholar 

  39. Schüttelkopf, A. W. & van Aalten, D. M. F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004)

    Article  PubMed  Google Scholar 

  40. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  PubMed  Google Scholar 

  41. Rocchia, W., Alexov, E. & Honig, B. Extending the applicability of the nonlinear Poisson–Boltzmann equation: multiple dielectric constants and multivalent ions. J. Phys. Chem. B 105, 6507–6514 (2001)

    Article  CAS  Google Scholar 

  42. Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 8, 127–134 (1995)

    Article  CAS  PubMed  Google Scholar 

  43. Irwin, J. J. et al. Automated docking screens: a feasibility study. J. Med. Chem. 52, 5712–5720 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Mysinger, M. M. & Shoichet, B. K. Rapid context-dependent ligand desolvation in molecular docking. J. Chem. Inf. Model. 50, 1561–1573 (2010)

    Article  CAS  PubMed  Google Scholar 

  45. Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kirchmair, J., Wolber, G., Laggner, C. & Langer, T. Comparative performance assessment of the conformational model generators Omega and Catalyst: a large-scale survey on the retrieval of protein-bound ligand conformations. J. Chem. Inf. Model. 46, 1848–1861 (2006)

    Article  CAS  PubMed  Google Scholar 

  47. Neudert, G. & Klebe, G. DSX: a knowledge-based scoring function for the assessment of protein-ligand complexes. J. Chem. Inf. Model. 51, 2731–2745 (2011)

    Article  CAS  PubMed  Google Scholar 

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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.), a Packard Foundation Fellowship (D.M.R.), an Emmy Noether Fellowship of the German Research Foundation (KO-4095/1-1 to P.K.) and COST Action GLISTEN (CM1207 to P.K.). We thank D. Borek and Z. Otwinowski for assistance with diffraction data processing. The National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility at the Advanced Photon Source is funded in whole or in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006).

Author information

Authors and Affiliations

Authors

Contributions

J.Y. expressed, purified and crystallized the hOX2R–PGS fusion protein, collected diffraction data, and solved the structure. J.C.M. performed computational docking experiments on synthetic orexin receptor antagonists. P.K. supervised and performed computational docking experiments. 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.

Corresponding author

Correspondence to Daniel M. Rosenbaum.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Purification of crystallization-grade hOX2R–PGS.

a, Superdex 200 gel filtration profile of hOX2R–PGS purified by nickel immobilized-metal affinity chromatography (Ni-IMAC) and M1-Flag immunoaffinity chromatography. b, Coommassie-stained polyacrylamide gel electrophoresis (PAGE) of the isolated peak fraction from gel filtration.

Extended Data Figure 2 Lipidic cubic phase crystallization setup for hOX2R–PGS.

The image shows representative microcrystals of the hOX2R–PGS protein that were harvested to produce high-resolution diffraction.

Extended Data Figure 3 Electron density map for suvorexant and surrounding residues.

The 2Fo − Fc electron density map is contoured at 1.2σ.

Extended Data Figure 4 Sequence alignment between hOX2R and hOX1R.

Positions that are identical between the two receptors are highlighted with a red background.

Extended Data Figure 5 Conservation of the orthosteric binding pocket between hOX2R and hOX1R.

Structure of the extracellular region of hOX2R, with residues that are identical between hOX2R and hOX1R coloured red, and residues that are different coloured grey. T1112.61 (to Ser) and T1353.33 (to Ala) are the only residues within 6 Å of suvorexant that are different between the two GPCRs. ECL3 is removed for clarity.

Extended Data Figure 6 Alternative docked poses for almorexant and EMPA.

a, Left, chemical structure of almorexant. Right, second docked pose of almorexant (green carbons) that was favourably scored and in agreement with mutational data. b, Left, chemical structure of EMPA. Right, second docked pose of EMPA (cyan carbons) that was favourably scored and in agreement with mutational data.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains descriptions of docking poses in Figure 3 and Extended Data Figure 6. (PDF 87 kb)

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Yin, J., Mobarec, J., Kolb, P. et al. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant. Nature 519, 247–250 (2015). https://doi.org/10.1038/nature14035

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