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Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gα s βγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gα s . Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function. Volta phase-plate cryo-electron microscopy reveals the structure of the full-length calcitonin receptor in complex with its peptide ligand and Gα s βγ. GPCR structure solved by cryo-electron microscopy The use of cryo-electron microscopy (cryo-EM) in structural biology has exploded in recent years as it provides structural information at near atomic resolution without the need for crystallization. However, cryo-EM has typically been limited to proteins larger than 200 kDa because of issues with low contrast. Patrick Sexton and colleagues report the structure of the full-length calcitonin receptor in complex with its peptide ligand and Gα s βγ protein by Volta phase-plate single-particle cryo-EM. This is the first G-protein-coupled receptor (GPCR) structure to be solved at high resolution by cryo-EM, the first full-length class B GPCR reported and only the second in complex with the full heterotrimeric G protein. The structure shows the GPCR in the active state and reveals key information about the conformational changes associated with peptide agonist binding and G-protein coupling in class B receptors.

The structure of the glucagon-like peptide 1 receptor (GLP-1R) with its biased agonist exendin-P5 sheds light on both receptor activation and the mechanism of biased agonism. The basis of bias in class B GPCRs The glucagon-like peptide 1 receptor (GLP-1R) is a key target for the treatment of type 2 diabetes and obesity. Recently, biased agonists—ligands that selectively activate primarily one signalling pathway—have been identified. One of these, exendin-P5, is biased for G-protein signalling, increasing adipogenesis—the generation of fat cells—and is more effective at correcting hyperglycaemia than other agonists. Here, Patrick Sexton and colleagues report the structure of the human GLP-1R in complex with exendin-P5 and the G-protein heterotrimer determined from cryo-electron microscopy. Not only does this work reveal more detailed structural information about the receptor complex and its activation, it also proposes a basis for biased agonism, in the interactions with both the ligand and the G protein. The class B glucagon-like peptide-1 (GLP-1) G protein-coupled receptor is a major target for the treatment of type 2 diabetes and obesity 1 . Endogenous and mimetic GLP-1 peptides exhibit biased agonism—a difference in functional selectivity—that may provide improved therapeutic outcomes 1 . Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gα s heterotrimer, determined at a global resolution of 3.3 Å. At the extracellular surface, the organization of extracellular loop 3 and proximal transmembrane segments differs between our exendin-P5-bound structure and previous GLP-1-bound GLP-1 receptor structure 2 . At the intracellular face, there was a six-degree difference in the angle of the Gαs–α5 helix engagement between structures, which was propagated across the G protein heterotrimer. In addition, the structures differed in the rate and extent of conformational reorganization of the Gα s protein. Our structure provides insights into the molecular basis of biased agonism.

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains. X-ray crystal structures of the M1 and M4 muscarinic acetylcholine receptors, revealing differences in the orthosteric and allosteric binding sites that help to explain the subtype selectivity of drugs targeting this family of receptors. M1 and M4 muscarinic acetylcholine receptor structures Arthur Christopoulos and colleagues present the first X-ray crystal structures of the M1 and M4 muscarinic acetylcholine receptors, G-protein-coupled receptors (GPCRs) that regulate many vital functions of the central and peripheral nervous systems. The structures reveal differences in the orthosteric and allosteric binding sites that help to explain the subtype selectivity of drugs targeting this family of receptors. The M1 and M4 receptor subtypes are potential drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia.

The adenosine A 1 receptor (A 1 R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain 1 , 2 . However, development of analgesic orthosteric A 1 R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects 3 . Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A 1 R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A 1 R co-bound to adenosine, MIPS521 and a G i2 heterotrimer, revealing an extrahelical lipid–detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine–receptor–G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts. MIPS521, a positive allosteric modulator of the adenosine A 1 receptor, has analgesic properties in a rat model of neuropathic pain through a mechanism by which MIPS521 stabilizes the complex between adenosine, receptor and G protein.

G protein–coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11–GPCR complexes, the Gq–α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from “unwinding” of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

The neuropeptide substance P (SP) is important in pain and inflammation. SP activates the neurokinin-1 receptor (NK1R) to signal via Gq and Gs proteins. Neurokinin A also activates NK1R, but leads to selective Gq signaling. How two stimuli yield distinct G protein signaling at the same G protein-coupled receptor remains unclear. We determined cryogenic-electron microscopy structures of active NK1R bound to SP or the Gq-biased peptide SP6–11. Peptide interactions deep within NK1R are critical for receptor activation. Conversely, interactions between SP and NK1R extracellular loops are required for potent Gs signaling but not Gq signaling. Molecular dynamics simulations showed that these superficial contacts restrict SP flexibility. SP6–11, which lacks these interactions, is dynamic while bound to NK1R. Structural dynamics of NK1R agonists therefore depend on interactions with the receptor extracellular loops and regulate G protein signaling selectivity. Similar interactions between other neuropeptides and their cognate receptors may tune intracellular signaling.Determination of the cryo-EM structures of active neurokinin-1 receptor bound to substance P or the Gq biased peptide SP6–11 reveals that interactions with the receptor extracellular loops regulate G protein signaling selectivity.

The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions, offering the potential for new drug treatments. However, progress in this area has been hindered by limited structural information and a lack of well-characterised tool compounds. In this study, we employ cryogenic electron microscopy (cryo-EM) to elucidate the structures of the P2X1 receptor in an ATP-bound desensitised state and an NF449-bound closed state. NF449, a potent P2X1 receptor antagonist, engages the receptor distinctively, while ATP, the endogenous ligand, binds in a manner consistent with other P2X receptors. To explore the molecular basis of receptor inhibition, activation, and ligand interactions, key residues involved in ligand and metal ion binding were mutated. Radioligand binding assays with [ 3 H]-α,β-methylene ATP and intracellular calcium ion influx assays were used to evaluate the effects of these mutations. These experiments validate key ligand-receptor interactions and identify conserved and non-conserved residues critical for ligand binding or receptor modulation. This research expands our understanding of the P2X1 receptor structure at a molecular level and opens new avenues for in silico drug design targeting the P2X1 receptor. The P2X1 receptor is a promising target for the development of a non-hormonal male contraceptive. Here, researchers determined the atomic structure of the P2X1 receptor, revealing opportunities for developing drugs to target this receptor.

The M 4 muscarinic acetylcholine receptor (M 4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M 4 mAChR in complex with the heterotrimeric G i1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M 4 mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M 4 mAChR. These findings provide a basis for further understanding xanomeline’s complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs. The drug Xanomeline is progressing through clinical trials for the treatment of patients with schizophrenia. Here, the authors determine a cryo-EM structure of Xanomeline bound to its primary target revealing a dual binding mode mechanism.

G-protein-coupled receptors (GPCRs) are key cell-surface proteins that transduce external environmental cues into biochemical signals across the membrane. GPCRs are intrinsically allosteric proteins; they interact via spatially distinct yet conformationally linked domains with both endogenous and exogenous proteins, nutrients, metabolites, hormones, small molecules and biological agents. Here we explore recent high-resolution structural studies, which are beginning to unravel the atomic details of allosteric transitions that govern GPCR biology, as well as highlighting how the wide diversity of druggable allosteric sites across these receptors present opportunities for developing new classes of therapeutics. High-resolution structural studies of GPCRs have led to insights into the role of allostery in GPCR-mediated signal transduction.

Adenosine receptors (ARs: A 1 AR, A 2A AR, A 2B AR, and A 3 AR) are crucial therapeutic targets; however, developing selective, efficacious drugs for them remains a significant challenge. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of the human A 3 AR in three distinct functional states: bound to the endogenous agonist adenosine, the clinically relevant agonist Piclidenoson, and the covalent antagonist LUF7602. These structures, complemented by mutagenesis and pharmacological studies, reveal an A 3 AR activation mechanism that involves an extensive hydrogen bond network from the extracellular surface down to the orthosteric binding site. In addition, we identify a cryptic pocket that accommodates the N 6 -iodobenzyl group of Piclidenoson through a ligand-dependent conformational change of M174 5.35 . Our comprehensive structural and functional characterisation of A 3 AR advances our understanding of adenosine receptor pharmacology and establishes a foundation for developing more selective therapeutics for various disorders, including inflammatory diseases, cancer, and glaucoma. The A3 adenosine receptor is a promising drug target for cancer, inflammation, and glaucoma. Here, authors determine atomic structures of the human A3 receptor, identifying a previously hidden binding pocket that will aid in the development of more effective A3 receptor-targeted medicines.