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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.

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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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