G protein-coupled receptors (GPCRs) are membrane proteins that act as signal transducers across cell membranes. Class B1 GPCRs, a subset of 15 receptors activated by peptide hormones, are involved in important physiological processes and diseases, making them a popular target for drug development. GPCRs are dynamic proteins and can adopt a myriad of conformational states, allowing them to bind and activate multiple intracellular signal transducers, including G proteins and β-arrestins, though all class B1 GPCRs primarily couple to the stimulatory G protein (Gs). Cryogenic electron microscopy (cryo-EM) structures of all class B1 GPCRs bound to Gs are available and provide meaningful insights into receptor function. However, there is a dearth of structural information on class B1 GPCRs in inactive and intermediate states or bound to other signal transducers, meaning we are currently only afforded a small vista into the conformational landscape these GPCRs sample. As cryo-EM-based 3D reconstructions are heavily dependent on protein stability and conformational homogeneity, currently available structures are largely limited to only those most stable conformations (i.e. Gs complexes). The present review focuses on technical aspects of obtaining class B1 GPCR structures using cryo-EM and new in silico methods that allow insight into unseen GPCR conformations, revealing more structural details of the conformational landscape.

G protein-coupled receptors (GPCRs) are key regulators of intercellular communication. One sub-family, named class B1 GPCRs, including the pituitary adenylate cyclase-activating polypeptide 1 receptor (PAC1R), is important for metabolic, cardiovascular, and endocrine functions. Currently, there is a wealth of structural data describing the active G protein-coupled state of class B1 GPCRs, and more limited information on inactive or intermediate states. Although these structures provide insight into conformational differences between different activation states, the receptor dynamics underpinning them remain largely unknown. Here, we employed hydrogen deuterium exchange mass spectrometry (HDX-MS) to investigate the dynamics of PAC1R across three distinct states: inactive (apo), intermediate (peptide agonist-bound), and fully active (peptide agonist and G protein-bound). This revealed dynamics of different states along the PAC1R activation pathway and deepens the understanding of class B1 GPCR dynamics and molecular mechanisms of receptor activation.

Abstract Positive allosteric modulators (PAMs) of the M 4 muscarinic acetylcholine receptor (mAChR) represent a promising therapeutic strategy for treating cognitive deficits and neuropsychiatric disorders. While first-generation M 4 mAChR PAMs, like LY2033298, demonstrated proof-of-concept, second-generation compounds, such as MK-97, exhibit substantially improved potency and reduced species variability. Here we report the cryo-EM structure of the M 4 mAChR bound to the endogenous agonist, acetylcholine, and MK-97 at 2.7 Å resolution, revealing the molecular basis for improved M 4 mAChR PAM activity. MK-97 adopts a distinctive ‘boomerang’-shaped conformation within the extracellular-facing allosteric binding site, with a central pyridine vertex, a lower cyclopentylmethylpyrazole arm extending toward the floor of the orthosteric site, and an upper isoindolinone arm projecting toward extracellular loop 2 (ECL2). This extended binding mode establishes a distributed interaction network across transmembrane helices TM2, TM3, TM5, TM6, and TM7, with key contacts including a hydrogen bond with Y92 2.64 and a π-π stacking interaction with W435 7.35 . Integration of structural data, molecular dynamics simulations, and mutagenesis validation reveals that the high affinity of MK-97 derives from optimized engagement across all three binding regions rather than dependence on any single critical contact. Insights from comprehensive structure-activity relationship (SAR) studies provide a molecular framework for the rational design of next-generation M 4 mAChR PAMs with improved pharmacological properties. Graphical Abstract

Activation of thyroid-stimulating hormone receptor (TSHR) by autoantibodies is the primary cause of the autoimmune diseases Graves' hyperthyroidism and Graves' ophthalmopathy. Thus, recent pharmaceutical research has focused on blocking pathogenic TSHR activation. While this has potential to offer first-in-class disease-modifying medications to treat Graves' disease, the success of targeting drugs requires understanding of how TSHR-stimulating autoantibodies activate the receptor and mediate signalling. This study comprehensively characterized in vitro pharmacological profiles of two human TSHR-stimulating autoantibodies, M22 and K1-18, relative to bovine TSH (bTSH) in a HEK293A cell background expressing recombinant human TSHR. M22 and K1-18 exhibited subtle, G protein-specific, differences in activating Gα proteins, with both antibodies generally less potent and/or efficacious than bTSH. While both antibodies were equivalent in activation of most G proteins, K1-18 was less potent than M22 in activation of Gαs-short and Gαz. Interestingly, bTSH, M22 and K1-18 more potently activated Gα15 compared to other Gα proteins. In addition, M22 and K1-18 activated Gαs-dependent cAMP production and Gαq-coupled signalling (IP1 accumulation and iCa2+ mobilisation), recruited β-arrestin 1 and 2, and stimulated receptor internalization to early endosomes. K1-18 was less potent than bTSH in all these assays, while M22 was less potent than bTSH in iCa2+ mobilisation and β-arrestin recruitment. While M22 and K1-18 largely mirror the pharmacology of bTSH, quantification of revealed relative to bTSH they exhibit bias towards Gα12 activation, and away from β-arrestin 2 recruitment and iCa2+ mobilization relative to Gs and cAMP. Thus, these findings offer insight into the action of TSHR-stimulating antibodies.

Receptor activity-modifying proteins (RAMPs) are critical modulators of class B1 G protein-coupled receptors (GPCRs), altering receptor pharmacology, trafficking, and signaling. The calcitonin receptor (CTR) forms heterodimers with each of the three RAMPs to generate amylin receptors (AMYRs) with distinct agonist selectivity and signaling profiles. Although recent cryo-electron microscopy (cryoEM) structures have advanced our understanding of AMYR architecture in fully active states, the dynamic and mechanistic basis of RAMP-dependent modulation of the CTR remains poorly understood. Here, we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe the conformational dynamics of the CTR alone and in complex with each RAMP in the apo (ligand-free) state. Our results reveal that RAMPs differentially influence the flexibility of key CTR domains, including the extracellular domain, transmembrane helices, and intracellular regions involved in G protein engagement. Furthermore, the RAMPs exhibit subtype-specific dynamic signatures, particularly within their transmembrane and C-terminal regions. Together, these findings reveal how RAMPs allosterically shape CTR conformational landscapes, providing a dynamic framework that links insights from static structural models to functional pharmacology.

G protein-coupled receptors (GPCRs) mediate information transfer to cells from the surrounding environment. In most cases, signaling is initiated or amplified when the receptor binds to an agonist, an event that alters the conformational profile of the receptor. Signal transduction results from interaction between the agonist-receptor complex and cytosolic partners such as G proteins, GPCR kinases (GRKs), and β-arrestins. Changes in agonist structure can lead to "signal bias", i.e., changes in the relative strength of signaling involving different partners. Some GPCRs, including those activated by long peptide hormones, continue to signal after internalization. In these cases, changes in agonist structure can lead to changes in the relative extent of signaling from different sites, e.g., cell surface vs endosomes ("location bias"). Many GPCRs are targets of approved drugs or drug candidates, and tuning signal bias and/or location bias is widely considered to be important for optimizing therapeutic profiles. Here we report another mechanism of modulating outcome via agonist modification: alteration of intracellular trafficking. The synthetic peptide agonist designated SPT, which contains five β-amino acid residues, was previously shown to activate the parathyroid hormone receptor-1 (PTH1R) and cause prolonged signaling in mice by an unknown mechanism. The SPT-PTH1R complex continues to stimulate cAMP production after internalization. We now find that the SPT-PTH1R complex impairs the sorting of early endosomes into recycling endosomes relative to the receptor complexed to the drug teriparatide. These findings suggest that altering intracellular GPCR trafficking patterns represents an unappreciated strategy for achieving prolonged action in vivo.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable, with GLP1R gene variation potentially linked to therapeutic outcomes. A GLP1R natural missense variant, A316T, protects against T2D and cardiovascular disease. Here, we generated and characterized a human GLP1R A316T mouse model. Human GLP1RA316T/A316T mice displayed lower fasting blood glucose versus wild-type littermates even under metabolic stress, as well as slower weight gain and alterations in islet cytoarchitecture, glucagon secretion, and liver metabolism under a high-fat, high-sucrose diet. This was however associated with blunted responses to pharmacological GLP-1RAs in vivo. Further investigations in β cell models demonstrated that human GLP1R A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R-targeting therapies.

G-protein-coupled receptors (GPCRs) have key roles in physiology and are central targets for drug discovery and development1,2, but the design of protein agonists and antagonists has been challenging as GPCRs are integral membrane proteins and conformationally dynamic3-6. Here we describe de novo design methods and a high-throughput receptor-diversion microscopy-based screen for generating GPCR-binding miniproteins with high affinity, potency and selectivity. We design miniprotein agonists that activate receptors involved in itch and pain, as well as antagonists that inhibit receptors implicated in cancer, metabolic disorders such as diabetes and obesity, and migraines. The cryo-electron microscopy (cryo-EM) structures of five receptor-bound designs are close to the computational design models. A designed chemokine receptor antagonist mobilizes haematopoietic stem and progenitor cells in vivo at a level comparable to a clinically used drug, with fewer adverse effects.

Polymer nanodiscs are a research tool that allows membrane proteins (MPs) to be encapsulated by a surrounding amphipathic polymer, isolated and studied to understand their structural and physiological properties. An advantage of using polymer nanodiscs over other membrane mimetics can be found in their ability to natively solubilise membrane proteins (MPs) within an annulus of cellular phospholipids, however, potential polymer interactions with membrane constituents can hinder MP activity making the selection of a suitable polymer critical. This work demonstrates the native solubilisation of G-protein coupled A2A adenosine receptor (A2AR) by polymers with alternating units and cationic charge, poly(N-methyl-4-vinyl pyridinium iodide-co-N-alkyl-maleimides) (poly(MVP-co-AlkylMs)), and novel statistical copolymers with pseudozwitterionic charge, poly(potassium 3-sulfopropyl methacrylate-co-2-(trimethyl-amino) ethyl methacrylate-co-n-butyl methacrylate) (poly(KSPMA-co-TMAEMA-co-BMA)), both synthesised using RAFT polymerisation. After surveying a library of polymers within each class, A2AR extraction was the most efficient using poly(MVP-co-BM) (1 : 1 MVP : BM) and poly(KSPMA-co-TMAEMA-co-BMA) (1 : 1 : 1 KSPMA : TMAEMA : BMA). The optimal pH, temperature, solubilisation time, polymer concentration and ionic strength conditions required for extracting A2AR were identified and enabled a large-scale A2AR-nanodisc preparation. The yield of A2AR-poly(MVP-co-BM) was superior to A2AR-poly(KSPMA-co-TMAEMA-co-BMA) nanodiscs after affinity purification. Functional assessment of the reconstituted receptors was undertaken using fluorescence correlation spectroscopy (FCS) to determine the ligand binding capacity of A2AR stabilised within an alternating cationic poly(MVP-co-BM). These native nanodiscs retained their ability to specifically bind A2AR ligand antagonists.

G protein-coupled receptors (GPCRs) play key roles in physiology and are central targets for drug discovery and development, yet the design of protein agonists and antagonists has been challenging as GPCRs are integral membrane proteins and conformationally dynamic. Here we describe computational de novo design methods and a high throughput "receptor diversion" microscopy-based screen for generating GPCR binding miniproteins with high affinity, potency and selectivity, and the use of these methods to generate MRGPRX1 agonists and CXCR4, GLP1R, GIPR, GCGR and CGRPR antagonists. Cryo-electron microscopy data reveals atomic-level agreement between designed and experimentally determined structures for CGRPR-bound antagonists and MRGPRX1-bound agonists, confirming precise conformational control of receptor function. Our de novo design and screening approach opens new frontiers in GPCR drug discovery and development.

Obesity is a major and increasingly prevalent chronic metabolic disease with numerous comorbidities. While recent incretin-based therapies have provided pharmaceutical inroads into treatment of obesity, there remains an ongoing need for additional medicines with distinct modes of action as independent or complementary therapeutics. Among the most promising candidates, supported by phase 1 and 2 clinical trials, is cagrilintide, a long-acting amylin and calcitonin receptor agonist. As such, understanding how cagrilintide functionally engages target receptors is critical for future development of this target class. Here, we determine structures of cagrilintide bound to Gs-coupled, active, amylin receptors (AMY1R, AMY2R, AMY3R) and calcitonin receptor (CTR) and compare cagrilintide interactions and the dynamics of receptor complexes with previously reported structures of receptors bound to rat amylin, salmon calcitonin or recently developed amylin-based peptides. These data reveal that cagrilintide has an amylin-like binding mode but, compared to other peptides, induces distinct conformational dynamics at calcitonin-family receptors that could contribute to its clinical efficacy.

The global prevalence of obesity is skyrocketing at an alarming rate, with recent data estimating that one-in-eight people are now living with the disease. Obesity is a chronic metabolic disorder that shares underlying pathophysiology with other metabolically-linked diseases such as type 2 diabetes mellitus, cardiovascular disease and diabetic cardiomyopathy. There is a distinct correlation between type 2 diabetes status and the likelihood of heart failure. Of note, there is an apparent sexual dimorphism, with women disproportionately affected with respect to the degree of severity of the cardiac phenotype of diabetic cardiomyopathy that results from diabetes. The current pharmacotherapies available for the attenuation of hyperglycaemia in type 2 diabetes are not always effective, and have varying degrees of efficacy in the setting of heart failure. Insulin can worsen heart failure prognosis whereas metformin, sodium-glucose cotransporter 2 inhibitors (SGLT2i) and more recently, glucagon-like peptide-1 receptor agonists (GLP-1RAs), have demonstrated cardioprotection with their administration. This review will highlight the advancement of incretin therapies for individuals with diabetes and heart failure and explore newly-reported evidence of the clinical usefulness of GLP-1R agonists in this distinct phenotype of heart failure.

Signal duration and subcellular location are emerging as important facets of G protein–coupled receptor (GPCR) function. The glucagon-like peptide-1 receptor (GLP-1R), a clinically relevant class B1 GPCR, stimulates production of the second messenger cyclic adenosine monophosphate (cAMP) upon activation by the native hormone, GLP-1. cAMP production continues after the hormone–receptor complex has been internalized via endocytosis. Here, we report GLP-1 analogues that induce prolonged signaling relative to GLP-1. A single β-amino acid substitution at position 18, with the residue derived from ( S , S )- trans -2-aminocyclopentanecarboxylic acid (ACPC), enhances signaling duration with retention of receptor endocytosis. Pairing ACPC at position 18 with a second substitution, α-aminoisobutyric acid (Aib) at position 16, abrogates endocytosis, but prolonged signaling is maintained. Prolonged signaling is sensitive to the structure of the β residue at position 18. Cryoelectron microscopy structures of two GLP-1 analogues bound to the GLP-1R:Gs complex suggest substantial alterations to bound peptide structure and dynamics compared to the GLP-1:GLP-1R:Gs complex. These structural findings strengthen an emerging view that agonist dynamics in the receptor-bound state influence signaling profiles. Our results advance understanding of the structural underpinnings of receptor activation and introduce tools for exploring the impact of spatiotemporal signaling profiles following GLP-1R activation.

Introduction and Objective: Survodutide (Survo) is a therapy undergoing Phase III studies to investigate its suitability for treating people living with obesity and MASH. Survo is a dual agonist that increases energy expenditure and decreases energy intake via activation of Glucagon Receptor (GCGR) and GLP-1 Receptor (GLP-1R), respectively, leading to up to 19% weight-loss, and 64.5% fibrosis reduction in F2-F3. We investigated the molecular basis of Survo’s dual agonism and how this differs from other GCGR and GLP-1R agonists. Methods: We employed in vitro assays such as cyclic adenosine mono phosphate (cAMP) production to compare Survo to the endogenous peptides GLP-1, glucagon (GCG), and oxyntomodulin (Oxy). Cryogenic electron microscopy (cryo-EM) structures of GCGR and GLP-1R in complex with Survo and G protein were determined at high resolution. Results: Survo is a 29 amino acid (AA) peptide based on GCG with pos 18, 20 and 23 swapped to GLP-1 and pos 16 swapped to exendin-4. Pos 24 and 27-29 were changed to other AA’s and pos 2 to the novel unnatural AA Ac4c. Survo induces cAMP production 4-fold less potent compared to native GLP-1 at GLP-1R (EC50: 20 vs 5 pM, respectively) and 22-times less potent compared to native GCG at GCGR (EC50: 108 vs 5 pM for Survo and GCG, respectively) while resembling the dual agonist Oxy at both receptors (EC50: 36 and 47 pM at GLP-1R and GCGR, respectively). Cryo-EM structures show similar binding poses for Survo compared to other peptide agonists, with differential AA’s showing interactions that explain the assay data. The lysine side chain at pos 24 links to a fatty diacid to improve plasma half-life. Removal of the linker and diacid increased potency at both receptors (EC50: 2 and 23 pM at GLP-1R and GCGR, respectively) with subtle changes in cryo-EM structures. Conclusion: The activation profile of Survo at GCGR and GLP-1R can be rationalized by structural evidence and the gained knowledge will inform therapeutic approaches in both obesity and MASH targeted treatment regimes. We further give insight into the effect of peptide lipidation. E. Yuliantie: None. P.N.H. Trinh: None. F. Bumbak: None. X. Zhang: None. Y. Jiang: None. Q. Ou: None. R. Ebenhoch: Employee; Boehringer-Ingelheim. D. Weichert: Employee; Boehringer-Ingelheim. H. Nar: Employee; Boehringer-Ingelheim. P.M. Sexton: Research Support; AstraZeneca, Pfizer Inc, Boehringer-Ingelheim, Novo Nordisk A/S, Astex Pharmaceuticals. D. Wootten: Research Support; Novo Nordisk A/S, Boehringer-Ingelheim, Pfizer Inc. Stock/Shareholder; Septerna Inc, Dacra Therapeutics. Research Support; AstraZeneca.

α- and β-calcitonin gene-related peptides (αCGRP and βCGRP, respectively), together with adrenomedullin (AM) and AM2 are endogenous agonists of the CGRP family of receptors; CGRP receptor (CGRPR), AM1 receptor (AM1R), and AM2 receptor (AM2R). The high sequence homology and similar tissue distribution of αCGRP and βCGRP suggests they have overlapping physiological roles in pain pathways, inflammation, and metabolism, but recent data indicate potential differences in the signaling capabilities of these peptides. However, a comprehensive pharmacological characterization of βCGRP activity, compared to αCGRP, AM, and AM2 across the three CGRP family receptors, is lacking. In this study, we assessed proximal G protein coupling/activation, cognate second messenger production, regulatory protein recruitment and receptor trafficking induced by αCGRP, βCGRP, AM, and AM2 at the CGRPR, AM1R, and AM2R. Our findings revealed a distinct profile of transducer and regulatory protein engagement induced by βCGRP compared to αCGRP across these receptors. The identification of differences in pharmacological profiles for αCGRP and βCGRP indicates that they may have more distinct physiological roles than previously appreciated and may assist in distinguishing the roles of these two peptides for exploitation in targeted drug design.

Membrane cholesterol modulates the ability of cholecystokinin to act at the type 1 cholecystokinin receptor (CCK1R), with elevated levels of this lipid that can be present in obesity, disrupting stimulus-activity coupling. This occurs via direct effect on a cholesterol association (cholesterol recognition/interaction amino acid consensus) motif within CCK1R, whereas an analogous motif is present in the type 2 cholecystokinin receptor (CCK2R) with no functional impact of cholesterol on that receptor. We recently demonstrated that surface residues surrounding this motif in these 2 G protein-coupled receptors could be exchanged as a group to convert their cholesterol sensitivity. We now evaluate each of these residues individually and use molecular dynamics to study cholesterol interactions with this site. These studies show that cholesterol occupies this motif in both CCK1R and CCK2R with similar dominant poses in both receptors in normal, as well as in elevated cholesterol environments. The dominant pose of cholesterol changes in both receptors with elevated cholesterol, shifting proximity from transmembrane segment (TM) 3 toward transmembrane segment 5. Of note, cholesterol residence time at this motif is longer for CCK2R than CCK1R and longer in high cholesterol conditions at both receptors. This suggests that the cholesterol sensitivity of CCK1R is likely a dynamic event mediated by short, transient cholesterol interaction with CCK1R under normal conditions. One residue in position 5.52 was critical for this cholesterol effect on CCK1R, despite the prediction that this is not due to direct interaction with cholesterol. Instead, the packing of hydrophobic residues around 5.52 is tighter in CCK1R than in CCK2R, likely affecting the position and movement of transmembrane segment 5 that are important for stimulus-activity coupling. Significance Statement Membrane cholesterol can affect the function of select G protein-coupled receptors, such as the type 1 cholecystokinin receptor, that are targets for drugs in disease states in which membrane cholesterol can be elevated. This article studied the molecular basis for high cholesterol disruption of stimulus-activity coupling at this receptor using site-directed mutagenesis and molecular dynamics. The findings demonstrate that cholesterol residence time at a key cholesterol association motif within CCK1R is responsible for this effect.