RESUMEN
G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.
Asunto(s)
Proteínas de Unión al GTP/química , Guanosina Trifosfato/farmacología , Receptores de Calcitonina/agonistas , Receptores de Calcitonina/química , Adenosina Difosfato/biosíntesis , Animales , Células COS , Chlorocebus aethiops , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ligandos , Conformación Proteica , Multimerización de Proteína , Receptores de Calcitonina/metabolismoRESUMEN
Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.
Asunto(s)
Receptores de Hormona Liberadora de Corticotropina/ultraestructura , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/ultraestructura , Secuencia de Aminoácidos , Microscopía por Crioelectrón/métodos , Encefalinas , Humanos , Ligandos , Modelos Moleculares , Péptidos , Precursores de Proteínas , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructura , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Transducción de SeñalRESUMEN
Peptide drugs targeting class B1 G-protein-coupled receptors (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 signaling 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, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 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 CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Animales , Sitios de Unión/fisiología , Microscopía por Crioelectrón/métodos , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/química , Humanos , Péptidos/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Isoquinolinas/farmacología , Fenilalanina/análogos & derivados , Piridinas/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Receptor del Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Isoquinolinas/química , Cinética , Modelos Moleculares , Fenilalanina/química , Fenilalanina/farmacología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Piridinas/química , Homología Estructural de ProteínaRESUMEN
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 Gs-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.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina/ultraestructura , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Proteína 1 Modificadora de la Actividad de Receptores/ultraestructura , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/ultraestructura , Sitios de Unión , Péptido Relacionado con Gen de Calcitonina/química , Proteína Similar al Receptor de Calcitonina/química , Proteína Similar al Receptor de Calcitonina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Humanos , Simulación de Dinámica Molecular , Dominios Proteicos , Estabilidad Proteica , Proteína 1 Modificadora de la Actividad de Receptores/química , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/química , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, 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 A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound ß2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.
Asunto(s)
Adenosina/química , Adenosina/metabolismo , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Receptor de Adenosina A1/química , Receptor de Adenosina A1/ultraestructura , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Modelos Moleculares , Receptor de Adenosina A1/metabolismo , Rotación , Especificidad por SustratoRESUMEN
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 Gαs 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 Gαs protein. Our structure provides insights into the molecular basis of biased agonism.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/ultraestructura , Sitios de Unión , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/química , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
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 Gαsßγ 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 Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gß subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP Heterotriméricas/ultraestructura , Receptores de Calcitonina/clasificación , Receptores de Calcitonina/ultraestructura , Sitios de Unión , Membrana Celular/metabolismo , Secuencia Conservada , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Modelos Moleculares , Conformación Proteica , Receptores de Calcitonina/agonistas , Receptores de Calcitonina/metabolismoRESUMEN
Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-Gs complex and compared this structure to our recently published structure of the GCGR-Gs complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.
Asunto(s)
Microscopía por Crioelectrón , Péptidos/química , Receptores de Glucagón , Animales , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/ultraestructura , Humanos , Dominios Proteicos , Estructura Cuaternaria de Proteína , Receptores de Glucagón/agonistas , Receptores de Glucagón/química , Receptores de Glucagón/ultraestructuraRESUMEN
G protein-coupled receptors (GPCRs) can be differentially activated by ligands to generate multiple and distinct downstream signaling profiles, a phenomenon termed biased agonism. The glucagon-like peptide-1 receptor (GLP-1R) is a class B GPCR and a key drug target for managing metabolic disorders; however, its peptide agonists display biased signaling that affects their relative efficacies. In this study, we combined mutagenesis experiments and mapping of surface mutations onto recently described GLP-1R structures, which revealed two major domains in the GLP-1/GLP-1R/Gs protein active structure that are differentially important for both receptor quiescence and ligand-specific initiation and propagation of biased agonism. Changes to the conformation of transmembrane helix (TM) 5 and TM 6 and reordering of extracellular loop 2 were essential for the propagation of signaling linked to cAMP formation and intracellular calcium mobilization, whereas ordering and packing of residues in TMs 1 and 7 were critical for extracellular signal-regulated kinase 1/2 (pERK) activity. On the basis of these findings, we propose a model of distinct peptide-receptor interactions that selectively control how these different signaling pathways are engaged. This work provides important structural insight into class B GPCR activation and biased agonism.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos/farmacología , Animales , Células CHO , Calcio/metabolismo , Cricetulus , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Descubrimiento de Drogas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptor del Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/genética , Humanos , Ligandos , Modelos Moleculares , Mutagénesis , Péptidos/metabolismo , Fosforilación , Conformación Proteica , Dominios ProteicosRESUMEN
Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.
Asunto(s)
Receptor Muscarínico M4 , Receptores Muscarínicos , Humanos , Acetilcolina/metabolismo , Regulación Alostérica , Sitio Alostérico , Microscopía por Crioelectrón , Ligandos , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/metabolismoRESUMEN
Class B1 G protein-coupled receptors (GPCRs) play important roles in human physiology and disease pathology. Using cryo-electron microscopy (cryo-EM) and X-ray crystallography, the 3D structures of all 15 members of this receptor subfamily have been determined in recent years at the near-atomic level. Although they share many structural commonalities, they show distinct features in terms of ligand recognition and receptor activation. In-depth structural analyses have yielded valuable insights into the N termini of both peptide hormones and cognate receptors, the outward movement of transmembrane helix 6 (TM6), the allosteric modulation sites located in the transmembrane domain (TMD), and the constitutive signaling bias mediated by receptor splice variants. These provide new directions for the design of better therapeutic agents, thereby making these targets more druggable.
Asunto(s)
Receptores Acoplados a Proteínas G , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Ligandos , Dominios Proteicos , Receptores Acoplados a Proteínas G/químicaRESUMEN
The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists.
Asunto(s)
Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Péptido Intestinal Vasoactivo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Unión Proteica , Simulación de Dinámica MolecularRESUMEN
Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.
Asunto(s)
Agonistas de los Receptores de Amilina/química , Receptores de Polipéptido Amiloide de Islotes Pancreáticos/química , Animales , Microscopía por Crioelectrón , Humanos , Fenotipo , Conformación Proteica , Multimerización de Proteína , SalmónRESUMEN
The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we combine cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding to four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data reveal that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists.
Asunto(s)
Exenatida/química , Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/química , Oxintomodulina/química , Regulación Alostérica , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Exenatida/genética , Exenatida/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Células HEK293 , Humanos , Cinética , Ligandos , Simulación de Dinámica Molecular , Mutación , Oxintomodulina/genética , Oxintomodulina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The glucagon-like peptide-1 receptor (GLP-1R) regulates insulin secretion, carbohydrate metabolism, and appetite and is an important target for treatment of type 2 diabetes and obesity. Multiple GLP-1R agonists have entered into clinical trials, with some, such as semaglutide, progressing to approval. Others, including taspoglutide, failed due to the high incidence of side effects or insufficient efficacy. GLP-1R agonists have a broad spectrum of signaling profiles, but molecular understanding is limited by a lack of structural information on how different agonists engage with the GLP-1R. Here, we report cryoelectron microscopy (cryo-EM) structures and cryo-EM 3D variability analysis of semaglutide- and taspoglutide-bound GLP-1R-Gs protein complexes. These reveal similar peptide interactions to GLP-1 but different motions within the receptor and bound peptides, providing insights into the molecular determinants of GLP-1R peptide engagement.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptor del Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos Similares al Glucagón/química , Péptidos Similares al Glucagón/metabolismo , Complejos Multiproteicos/metabolismo , Péptidos/química , Péptidos/metabolismo , Línea Celular , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Cryo-electron microscopy (cryo-EM) of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Pushing the performance boundaries of the technique requires quantitative knowledge about the contribution of multiple factors. Here, we present an in-depth analysis and optimization of the main experimental parameters in cryo-EM. We combined actual structural studies with methods development to quantify the effects of the Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. By using this information to carefully maximize the experimental performance, it is now possible to routinely determine GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps enables the building of better atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines given here are not limited to GPCRs and can be applied directly to other small proteins.
Asunto(s)
Microscopía por Crioelectrón/métodos , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Microscopía por Crioelectrón/instrumentación , Oro , Procesamiento de Imagen Asistido por ComputadorRESUMEN
G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors from insect cells to determine their cryo-electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/química , Receptores de Péptido Relacionado con el Gen de Calcitonina/química , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina/química , Línea Celular , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Ligandos , Modelos Moleculares , Mariposas Nocturnas , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteína 1 Modificadora de la Actividad de Receptores/química , Proteína 1 Modificadora de la Actividad de Receptores/metabolismoRESUMEN
Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AM1R and AM2R, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AM1R in complex with AM and Gs at a global resolution of 3.0 Å, and structures of the AM2R in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 Å, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.
RESUMEN
The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.