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.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins: insight into mechanisms of G protein selectivity

“…The transmembrane core of the active-state CCK1R is similar to other active-state class A GPCR structures (6) with the intracellular side of TM6 occupying a similar location to other active, Gq/11-coupled, receptors (23,24,27), creating a binding site for the insertion of the a5 helix of the G proteins. While there are no inactive-state structures available for the CCK1R, the rotameric positions of residues in conserved class A activation motifs such as the CWxP, PI(T)F, NPxxY, and E/DRY motifs exhibited strong overlap with the equivalent residues in the active OX2R complex (24) (Fig.…”

“…The high resolution of this structure enabled modelling of three water molecules within the CCK-8 binding pocket, one of which (H2O-2) is coordinated by the SO3 group of Y2-SO3 CCK , and also interacts with the backbone of M6 CCK and N98 2.61 of CCK1R. The location of CCK-8, which is deep within the TM binding pocket, is observed in other class A peptide-bound receptor structures, such as the OX2R, which also binds a C-terminally amidated peptide (OxB) (24). The similar depth of OxB within the OX2R binding pocket also enables hydrogen bonding to Y 7.43 .…”

“…In this study, we used cryo-electron microscopy to determine structures of the CCK1R bound to the CCK peptide agonist, CCK-8 and two different transducer proteins; GasGb1g2 and a chimeric Gq protein mimic (with Gb1g2) that was recently used to determine the structure of Gq/11 coupled 5HT2A and orexin 2 (OX2) receptors (23,24). As seen with other Gq/11-GPCR complexes, the Gq-a5 helix bound to a relatively narrow pocket in the CCK1R core.…”

“…In one approach, used with the muscarinic M1 receptor, chimeras of G11 with the aN of Gi were generated to enable the use of the short chain antibody, scFv16, to bridge this aN and the b-subunit of the obligate Gbg dimer (27). For the 5HT2A and OX2 receptors, further engineering was required (23,24). In this case a chimera of mini-Gs substituted with (i) Gq residues proximal to the receptor interface (including the Cterminal aH5) and (ii) the far aN of Gi (mGsqi; Fig.…”

“…The CCK1R is preferentially coupled to Gq/11 proteins. Only 3 other structures of GPCRs (5HT2AR, OX2R, M1 AChR) bound to Gq-mimetic proteins have been determined (23,24,27), where Gq or G11is also the primary transducer. In each of these receptors, TM6 remains in an extended helical conformation that is translated out at the intracellular base relative to inactive structures (23,24); the G protein a5 helix binds into the narrow cavity formed by this translational movement (Fig.…”