SUMMARY Class B G protein-coupled receptors are major targets for treatment of chronic diseases, including 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 Gαβγs protein determined by Volta phase plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor through 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 large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G protein coupling via interaction with the Gβ subunit. This structure provides a new framework for understanding G protein-coupled receptor function.

Allosteric ligands bind to G protein-coupled receptors (GPCRs; also known as seven-transmembrane receptors) at sites that are distinct from the sites to which endogenous ligands bind. The existence of allosteric ligands has enriched the ways in which the functions of GPCRs can be manipulated for potential therapeutic benefit, yet the complexity of their actions provides both challenges and opportunities for drug screening and development. Converging avenues of research in areas such as biased signalling by allosteric ligands and the mechanisms by which allosteric ligands modulate the effects of diverse endogenous ligands have provided new insights into how interactions between allosteric ligands and GPCRs could be exploited for drug discovery. These new findings have the potential to alter how screening for allosteric drugs is performed and may increase the chances of success in the development of allosteric modulators as clinical lead compounds.

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. Endogenous and mimetic GLP-1 peptides exhibit biased agonism-a difference in functional selectivity-that may provide improved therapeutic outcomes. Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gα 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. 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 protein. Our structure provides insights into the molecular basis of biased agonism.

G protein-coupled receptors (GPCRs) are the largest group of cell surface receptors in humans that signal in response to diverse inputs and regulate a plethora of cellular processes. Hence, they constitute one of the primary drug target classes. Progress in our understanding of GPCR dynamics, activation and signalling has opened new possibilities for selective drug development. A key advancement has been provided by the concept of biased agonism, which describes the ability of ligands acting at the same GPCR to elicit distinct cellular signalling profiles by preferentially stabilizing different active conformational states of the receptor. Application of this concept raises the prospect of 'designer' biased agonists as optimized therapeutics with improved efficacy and/or reduced side-effect profiles. However, this application will require a detailed understanding of the spectrum of drug actions and a structural understanding of the drug-receptor interactions that drive distinct pharmacologies. The recent revolution in GPCR structural biology provides unprecedented insights into ligand binding, conformational dynamics and the control of signalling outcomes. These insights, together with new approaches to multi-dimensional analysis of drug action, are allowing refined classification of drugs according to their pharmacodynamic profiles, which can be linked to receptor structure and predictions of preclinical drug efficacy.

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the G-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

The class A adenosine A receptor (AR) is a G-protein-coupled receptor that preferentially couples to inhibitory G heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human AR in complex with adenosine and heterotrimeric G protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive AR, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the AR primarily via amino acids in the C terminus of the Gα α5-helix, concomitant with a 10.5 Å outward movement of the AR transmembrane domain 6. Comparison with the agonist-bound β adrenergic receptor-G-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active AR structure provides molecular insights into receptor and G-protein selectivity.

Recently, the concept of ligand-directed signaling-the ability of different ligands of an individual receptor to promote distinct patterns of cellular response-has gained much traction in the field of drug discovery, with the potential to sculpt biological response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Here, we demonstrate the importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clinically used mimetic exendin-4.

Peptide drugs targeting class B1 GPCRs can treat multiple diseases, however there remains substantial interest in the development of orally delivered non-peptide drugs. Here we reveal unexpected overlap between signalling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist, PF 06882961, and GLP-1 that was not observed for another compound, OWL-833. Both compounds are currently in clinical trials for treatment of type 2 diabetes. High resolution cryo-EM structures reveal the binding sites for PF-06882961 and GLP-1 substantially overlap, whereas OWL-833 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not OWL-833, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of nonpeptide agonists targeting class B GPCRs.