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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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).
The authors declare no competing financial interests.
Extended data figures and tables
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.
The image shows representative microcrystals of the hOX2R–PGS protein that were harvested to produce high-resolution diffraction.
The 2Fo − Fc electron density map is contoured at 1.2σ.
Positions that are identical between the two receptors are highlighted with a red background.
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.
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.
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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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