ARTICLE https://doi.org/10.1038/s41467-021-21087-6 OPEN Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation Chuan Hong 1,8, Noel J. Byrne 2,8, Beata Zamlynny3, Srivanya Tummala 2, Li Xiao 1, Jennifer M. Shipman2, Andrea T. Partridge 2, Christina Minnick 4, Michael J. Breslin5, Michael T. Rudd 5, Shawn J. Stachel 5, Vanessa L. Rada 5, Jeffrey C. Kern5, Kira A. Armacost2, Scott A. Hollingsworth 6, Julie A. O’Brien4, Dawn L. Hall2, Terrence P. McDonald7, Corey Strickland1, Alexei Brooun2, ✉ ✉ Stephen M. Soisson 2 & Kaspar Hollenstein 2 1234567890():,; 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. 1 Computational & Structural Chemistry, MRL, Merck & Co., Inc, Kenilworth, NJ, USA. 2 Computational & Structural Chemistry, MRL, Merck & Co., Inc, West Point, PA, USA. 3 Screening & Compound Profiling, MRL, Merck & Co., Inc, Kenilworth, NJ, USA. 4 Quantitative Bioscience, MRL, Merck & Co., Inc, West Point, PA, USA. 5 Discovery Chemistry, MRL, Merck & Co., Inc, West Point, PA, USA. 6 Computational & Structural Chemistry, MRL, Merck & Co., Inc., South San Francisco, CA, USA. 7 Neuroscience, MRL, Merck & Co., Inc, West Point, PA, USA. 8These authors contributed equally: Chuan Hong, Noel J. Byrne. ✉ email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:815 | https://doi.org/10.1038/s41467-021-21087-6 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21087-6 β γ rexin A (OxA) and orexin B (OxB), also termed presence of OxB and assembled a complex with mini-Gsqi,G 1 2, Ohypocretin-1 and hypocretin-2, respectively, are excitatory and scFv16. Samples were vitrified on electron microscopy grids neuropeptides produced in the hypothalamus that control and the structure was determined by single-particle cryo-EM to a sleep/wake behavior by promoting and maintaining wakefulness, nominal resolution of 3.2 Å (Supplementary Table 1 and Sup- and suppressing rapid eye movement (REM) sleep1–3.Lossof plementary Fig. 2). The resulting density map was of high quality orexin-producing neurons is the cause of narcolepsy type 1 (NT1), and allowed modeling of sidechains for most of the amino acids also termed classical narcolepsy or narcolepsy with cataplexy4,5,a of the complex (Supplementary Fig. 3). Residues N20–M28 at the lifelong neurological disorder that affects 1 in 2000 to 1 in 4000 carboxy-terminus of OxB were well-resolved, while only poor or people6–8. Patients suffer from excessive daytime sleepiness and no density for the remainder of the peptide was observed. We abnormal REM sleep-related symptoms, such as hallucinations, obtained improved density maps at an overall resolution of 3.0 Å sleep paralysis, and cataplexy. Available therapies are limited to for the structure with compound 1 (Fig. 1a) by including a syn- managing symptoms with modest effectiveness and moderate to thetic nanobody, Sb51, for additional conformational stabilization severe adverse effects9. and to aid alignment of particle projections (Supplementary OxA and OxB signal through the two closely related receptors, Table 1, and Supplementary Figs. 4 and 5). Sb51 binds to the orexin receptor type 1 and type 2 (OX1R and OX2R, respectively), extracellular surface of OX2R contacting residues in extracellular 3 expressed widely across the central nervous system .OX1R and loop (ECL) 2, and part of ECL3 without directly interacting with OX2R are class A G-protein-coupled receptors (GPCRs) of the β- compound 1 or significantly altering the structure of the small- – branch that signal predominantly through heterotrimeric Gq/11 molecule binding site, suggesting that the compound 1 receptor leading to increased cytosolic Ca2+ levels10. Inhibition of both interactions are likely unaffected by the presence of the nano- OX1R and OX2R with antagonists provides an effective treatment body. Sb51 was omitted, however, for the complex with OxB as it of insomnia with two drugs recently reaching the market11–14. was likely to clash with the much larger peptide agonist or Conversely, intracerebroventricular administration of OxA pro- indirectly affect the receptor–peptide interface. motes wakefulness and suppresses REM sleep in mice15 and, more recently, the discovery of non-peptide orexin agonists have Overall structures of active-state OX R. The structures of OX R demonstrated potential for the treatment of NT1 and other 2 2 – bound to OxB and compound 1 are very similar, with root mean hypersomnia disorders16 18, by targeting the orexin system via square deviations of 0.77 and 0.54 Å, when comparing equivalent α- selective activation of OX R. Despite progress with two OX R- 2 2 carbons of OX R and of the entire receptor–G-protein complexes, selective agonists in early clinical trials19, identification of effi- 2 respectively (Supplementary Fig. 6). The main structural differences cacious oral small-molecule agonists with drug-like properties are in ECL2 and ECL3 of OX R, regions that contribute to the remains challenging. 2 epitope of Sb51. The structures of the nucleotide-free complexes Recently reported structures of antagonist-bound OX Rand – 1 and the relative orientation of the individual subunits closely OX R20 23 have greatly advanced our understanding of the mole- 2 resemble previously reported structures of GPCRs bound to G cular basis of antagonist recognition and subtype selectivity. Yet, – proteins G ,G,G,orG (refs. 25,27 30), and are consistent with their utility for dissecting the molecular mechanism of activation i o s 11 what has been referred to as a canonical-state complex31 (Fig. 1c, d). and for driving the discovery of small-molecule agonists has Comparison of the OxB-bound structure with the structures of remained limited. In this work, we determined single-particle cryo- inactive-state OX R reveals the conformational changes the receptor electron microscopy (cryo-EM) structures of OX R–G-protein 2 2 undergoes upon agonist binding and activation (Fig. 2a–c). The complexes bound to OxB and the small-molecule agonist 3′-(N-(3- observed reorganization of transmembrane (TM) helices 5–7atthe (2-(2-(2H-1,2,3-triazol-2-yl)benzamido)ethyl)phenyl)sulfamoyl)-4′- cytosolic interface are consistent with the key conformational methoxy-N,N-dimethyl-[1,1′-biphenyl]-3-carboxamide (compound changes associated with the conserved mechanism of GPCR 1;Fig.1a) to further our understanding of orexin signaling and to activation32,33, and result in the intracellular half of TM6 swinging enable structure-based drug discovery of novel therapeutics for the outward to allow insertion of the α5 helix at the carboxy-terminus treatment of NT1. Our results shed light on the molecular details of of the Gα-subunit deep into the core of OX R. Furthermore, the peptide and small-molecule agonist recognition, reveal the global 2 arrangements of several structural motifs associated with GPCR and local conformational changes associated with receptor activa- activation suggest that the receptor has been visualized in a fully tion, and suggest a molecular mechanism for activation by peptide activated state (Fig. 2d). They include the microswitches R1523.50 and small-molecule agonists. (Ballesteros-Weinstein numbering in superscript34), and Y3647.50 of the D3.49R3.50Y3.51 motif and the N7.49P7.50xxY7.53 motif, respec- 3.40 5.50 6.44 Results tively, as well as the hydrophobic core triad I/V P F (also termed connector region). Structure determination. We reconstituted an OX R–G-protein 2 The rearrangements on the extracellular side are more subtle complex from purified subunits, using an engineered variant of and lead to the contraction of the solvent-accessible central cavity human OX R with a truncated carboxy-terminus and a shortened 2 of the receptor (Fig. 1c). Of note is that in the structures of intracellular loop (ICL) 3. This construct enabled high-level antagonist-bound OX R, TM2 is partially unwound and kinked heterologous expression and purification of homogeneous mate- 2 toward TM7 facilitated by conserved P1092.59, while in active- rial, while maintaining wild-type-like affinity for both the natural state OX R, the helical twist is tightened, causing an inward peptide agonists and the small-molecule agonist compound 1 2 displacement of residues on either side of the kink. Moreover, (Supplementary Fig. 1). We used a Gα-subunit based on a pre- enabled by sidechain rearrangements in the hydrophobic core of viously described chimeric minimal Gα-construct with altered fi OX2R, TM3 undergoes a minor rotation and moves along the receptor speci city to allow productive interaction with Gq-cou- 24 α α helical axis toward the extracellular side. In addition, the entire pled GPCRs . We included residues of the N helix of G i1 to α helix is slightly shifted toward the center of the helical bundle. obtain a G -construct we refer to as mini-Gsqi. The altered amino-terminus allowed the use of previously described antibody fragment scFv16 that stabilizes the nucleotide-free receptor–G- Peptide binding in the receptor core.