Crystal Structures of Human Orexin 2 Receptor Bound to the Subtype-Selective Antagonist EMPA

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX₂R structures in complex with selective antagonists and previously determined OX₁R/OX₂R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX₂R. The importance of these residues for binding selectivity to OX₂R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors. Molecular dynamics simulations and structural information for orexin receptors indicates that the residues at positions 2.61 and 3.33 are critical for antagonist selectivity. Suno et al. determine the structure of a human GPCR using SACLA, and characterize differences between XFEL and synchrotron.