Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation

Abstract

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX₂R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX₂R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX₂R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX₂R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX₂R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

Fig. 1. Overall structures of peptide agonist and small-molecule agonist-activated OX₂R.

a Structural formula of the small-molecule orexin agonist used in this study. b Concentration-dependent activation of OX₁R and OX₂R by OxB and compound 1. Half maximal effective concentrations represented as pEC₅₀ are: 4.45 ± 0.06 (OX₁R–compound 1), 8.28 ± 0.03 (OX₂R–compound 1), 6.09 ± 0.14 (OX₁R–OxB), and 7.17 ± 0.07 (OX₂R–OxB); error bars represent the standard error of the mean of n = 4 independent experiments for all except for OX₁R/compound 1, for which n = 5 independent experiments were performed. Data are presented as mean values ± s.e.m. c, d Structures of nucleotide-free OX₂R–G-protein complexes bound to OxB and compound 1, respectively, color-coded by subunit. Source data are provided as a Source Data file.

Fig. 2. Comparison of inactive-state and active-state OX₂R.

a Superposition of OxB-activated (green) and inactive-state OX₂R (gray; PDB ID 5WQC) viewed from two different angles from within the membrane. Red arrows indicate conformational transitions upon OX₂R activation. b, c Intracellular and extracellular view of the superimposed receptor structures, respectively. Residues 192–214 of ECL2 have been removed for clarity. d Arrangement of conserved motifs associated with receptor activation: DRY (left), NPxxY (middle), and hydrophobic core triad (right). Structures with OxB (green) and compound 1 (purple) are overlaid with inactive-state OX₂R (gray).

Fig. 3. Small-molecule and peptide agonist recognition.

a Electron density map (blue mesh) around residues N20–M28 of OxB (cyan) viewed from two angles. b, c Two different views of the detailed interactions of OxB and OX₂R (green). d Electron density map (blue mesh) around compound 1 (yellow) viewed from two angles. e Detailed interactions of compound 1 with OX₂R (purple). f Overlay of the binding sites with OxB and compound 1. Residues 192–214 of ECL2 have been removed for clarity. The asterisks in a, c, and f indicate the position of the mainchain nitrogen of N20 of OxB.

Fig. 4. Molecular dynamics simulations.

a, b Representative frames from a 1000-ns MD trajectory overlaid with the model of OX₂R bound to full-length OxB (sand). OX₂R and OxB ensembles from the simulation are colored gray and yellow, respectively. The amino-terminal α-helix and ECL2 of OX₂R are highlighted in cyan and light blue, respectively. Note, in a, OxB was omitted for clarity. c MD-derived density map (orange mesh) of OxB defining the space occupied by the peptide during the simulations, showing that the amino-terminal portion of OxB remains α-helical, but is more flexible than the extended carboxy-terminal portion buried in the OX₂R core. d, e Root mean square fluctuation (RMSF) values of α-carbons of OX₂R and OxB, respectively, during a representative simulation. f–h Distances between α-carbons of pairs of residues in OxB and OX₂R monitored over the course of the same representative simulation. Residue pairs are: Q12(OxB)–F197 (ECL2 of OX₂R), Q12(OxB)–E46 (amino-terminal helix of OX₂R), and I25(OxB)–F346^7.35 (core of OX₂R), respectively. A central moving average (window length: 10 ns) of the α-carbon distance for each pair is indicated by a dark blue line. Source data are provided as a Source Data file.

Fig. 5. Comparison of agonist and antagonist binding.

Superpositions of OX₂R in inactive (gray) and active (green and purple) conformations. a–c Comparison of the binding mode of compound 1 (yellow) and those of the antagonists EMPA (blue; PDB ID 5WQC), suvorexant (orange, 4S0V), and HTL6641 (dark green; 6TPN), respectively. Water molecules mediating interactions with the sidechain of H350^7.39 in the antagonist-bound complexes are rendered as red spheres. d–f Conformational changes (white arrows) in TM2, TM3, Q134^3.32, and C107^2.56 upon activation by OxB (cyan) and compound 1 are highlighted by white arrows.

Fig. 6. Mechanism of OX₂R activation and inactivation.

Schematic representation of OX₂R in ligand-free (middle), antagonist-bound (left), and agonist-bound (right) states. The sidechain of mechanistically important Q134^3.32 is shown in stick representation. Conformational flexibility in TM2, TM3, and TM6 in the ligand-free state is indicated with gray arrows, while conformational changes upon agonist and antagonist binding are highlighted by green and red arrows, respectively.