RESUMEN
G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsß1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gßγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/química , Receptor de Adenosina A2A/química , Regulación Alostérica , Sitios de Unión , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Cinética , Ligandos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Nanoestructuras/química , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transducción de SeñalRESUMEN
Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , beta-Arrestinas/metabolismo , Transferencia de Energía por Resonancia de Bioluminiscencia , AMP Cíclico/metabolismo , Endosomas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células HEK293 , Humanos , Microscopía Confocal , Microscopía Electrónica , Complejos Multiproteicos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , beta-Arrestinas/químicaRESUMEN
G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized ß2-adrenergic receptor (ß2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that ß2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated ß2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal ß2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.
Asunto(s)
Adrenérgicos , Endosomas , Quinasas MAP Reguladas por Señal Extracelular , Receptores Adrenérgicos beta 2 , Adrenérgicos/metabolismo , Adrenérgicos/farmacología , Proliferación Celular , Endosomas/efectos de los fármacos , Endosomas/enzimología , Endosomas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genes myc , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease1 and antipsychotic drugs2. DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine3, leading to stimulation of Gi and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-Gi complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular Gi-binding site. The DRD2-Gi structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.
Asunto(s)
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 , Lípidos de la Membrana/metabolismo , Membranas Artificiales , Receptores de Dopamina D2/química , Receptores de Dopamina D2/ultraestructura , Bromocriptina/química , Bromocriptina/metabolismo , Dopamina/química , Dopamina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Lípidos de la Membrana/química , Modelos Moleculares , Conformación Proteica , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Transducción de SeñalRESUMEN
Over the past five decades, tremendous effort has been devoted to computational methods for predicting properties of ligands-i.e., molecules that bind macromolecular targets. Such methods, which are critical to rational drug design, fall into two categories: physics-based methods, which directly model ligand interactions with the target given the target's three-dimensional (3D) structure, and ligand-based methods, which predict ligand properties given experimental measurements for similar ligands. Here, we present a rigorous statistical framework to combine these two sources of information. We develop a method to predict a ligand's pose-the 3D structure of the ligand bound to its target-that leverages a widely available source of information: a list of other ligands that are known to bind the same target but for which no 3D structure is available. This combination of physics-based and ligand-based modeling improves pose prediction accuracy across all major families of drug targets. Using the same framework, we develop a method for virtual screening of drug candidates, which outperforms standard physics-based and ligand-based virtual screening methods. Our results suggest broad opportunities to improve prediction of various ligand properties by combining diverse sources of information through customized machine-learning approaches.
Asunto(s)
Antipsicóticos/química , Antipsicóticos/farmacología , Diseño de Fármacos/métodos , Inteligencia Artificial , Sitios de Unión , Regulación de la Expresión Génica/efectos de los fármacos , Ligandos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Relación Estructura-ActividadRESUMEN
Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the ß2-adrenergic receptor (ß2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for ß2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/química , Antagonistas Adrenérgicos beta/química , Alprenolol/química , Norepinefrina/química , Receptores Adrenérgicos beta 2/química , Xinafoato de Salmeterol/química , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Antagonistas Adrenérgicos beta/farmacología , Regulación Alostérica , Sitio Alostérico , Alprenolol/farmacología , Células HEK293 , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Norepinefrina/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Receptores Adrenérgicos beta 2/metabolismo , Xinafoato de Salmeterol/farmacología , Termodinámica , Agua/químicaRESUMEN
G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the ß2-adrenergic receptor (ß2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the ß2AR stabilizes a 'closed' receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCRG-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.
Asunto(s)
Sitio Alostérico , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gs/farmacología , Guanina/metabolismo , Guanina/farmacología , Humanos , Cinética , Ligandos , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Receptores Adrenérgicos beta 2/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacologíaRESUMEN
In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.
RESUMEN
Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.
Asunto(s)
Receptor Muscarínico M3/antagonistas & inhibidores , Receptor Muscarínico M3/genética , Acetilcolina/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Simulación del Acoplamiento Molecular/métodos , Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/metabolismo , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M2/metabolismoRESUMEN
Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 µM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.
Asunto(s)
Agonistas Muscarínicos/química , Receptor Muscarínico M2/química , Regulación Alostérica , Sitio Alostérico , Animales , Humanos , Cinética , Ligandos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Simulación del Acoplamiento Molecular , Agonistas Muscarínicos/metabolismo , Fosforilación , Unión Proteica , Ratas , Receptor Muscarínico M2/metabolismoRESUMEN
Salmeterol is a partial agonist for the ß2 adrenergic receptor (ß2AR) and the first long-acting ß2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound ß2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between ß1AR and ß2AR explain the high receptor-subtype selectivity. A structural comparison with the ß2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited ß-arrestin recruitment for salmeterol.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/química , Xinafoato de Salmeterol/química , Animales , Anticuerpos/química , Asma/tratamiento farmacológico , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Proteínas de Unión al GTP/química , Humanos , Enlace de Hidrógeno , Ligandos , Lípidos/química , Mutagénesis , Unión Proteica , Conformación Proteica , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Transducción de Señal , beta-Arrestinas/químicaRESUMEN
The Frizzled (FZD) proteins belong to class F of G protein-coupled receptors (GPCRs) and are essential for various pathways involving the secreted lipoglycoproteins of the wingless/int-1 (WNT) family. A WNT-binding cysteine-rich domain (CRD) in FZDs is N-terminally located and connected to the seven transmembrane domain-spanning receptor core by a linker domain that has a variable length in different FZD homologs. However, the function and importance of this linker domain are poorly understood. Here we used systematic mutagenesis of FZD6 to define the minimal N-terminal domain sufficient for receptor surface expression and recruitment of the intracellular scaffold protein Dishevelled (DVL). Further, we identified a triad of evolutionarily conserved cysteines in the FZD linker domain that is crucial for receptor membrane expression and recruitment of DVL. Our results are in agreement with the concept that the conserved cysteines in the linker domain of FZDs assist with the formation of a common secondary structure in this region. We propose that this structure could be involved in agonist binding and receptor activation mechanisms that are similar to the binding and activation mechanisms known for other GPCRs.
Asunto(s)
Proteínas Dishevelled/metabolismo , Receptores Frizzled/metabolismo , Aciltransferasas/antagonistas & inhibidores , Bencenoacetamidas/farmacología , Sitios de Unión , Membrana Celular/metabolismo , Cisteína/química , Receptores Frizzled/genética , Células HEK293 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Mutagénesis , Mutación , Dominios Proteicos , Piridinas/farmacología , Proteínas Wnt/metabolismo , Vía de Señalización WntRESUMEN
A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the ß2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.
Asunto(s)
Técnicas Biosensibles/métodos , Endosomas/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Cubiertas por Clatrina , AMP Cíclico/metabolismo , Endocitosis , Endosomas/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Isoproterenol/farmacología , Modelos Biológicos , Conformación Proteica , Receptores Adrenérgicos beta 2/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.
Asunto(s)
Morfinanos/química , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Ratones , Modelos Moleculares , Morfinanos/metabolismo , Morfinanos/farmacología , Conformación Proteica , Multimerización de Proteína , Receptores Opioides mu/metabolismo , Solventes/químicaRESUMEN
Cell transmembrane receptors play a key role in the detection of environmental stimuli and control of intracellular communication. G protein-coupled receptors constitute the largest transmembrane protein family involved in cell signaling. However, current methods for their functional reconstitution in biomimetic membranes remain both challenging and limited in scope. Herein, we describe the spontaneous reconstitution of adenosine A2A receptor (A2AR) during the de novo formation of synthetic liposomes via native chemical ligation. The approach takes advantage of a nonenzymatic and chemoselective method to rapidly generate A2AR embedded phospholiposomes from receptor solubilized in n-dodecyl-ß-d-maltoside analogs. In situ lipid synthesis for protein reconstitution technology proceeds in the absence of dialysis and/or detergent absorbents, and A2AR assimilation into synthetic liposomes can be visualized by microscopy and probed by radio-ligand binding.
Asunto(s)
Liposomas/metabolismo , Receptor de Adenosina A2A/metabolismo , Humanos , Liposomas/síntesis química , Liposomas/química , Modelos Moleculares , Estructura Molecular , Receptor de Adenosina A2A/químicaRESUMEN
G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human ß(2) adrenergic receptor (ß(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the ß(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the ß(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the ß-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and ß-phosphate coordination are key determinants of GDP (and GTP) binding affinity.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animales , Biocatálisis , Dominio Catalítico , Bovinos , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores Adrenérgicos beta 2/ultraestructuraRESUMEN
G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human ß(2) adrenergic receptor (ß(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive ß(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/inmunología , Nanoestructuras/química , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/inmunología , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animales , Sitios de Unión , Camélidos del Nuevo Mundo , Cristalografía por Rayos X , Agonismo Inverso de Drogas , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/farmacología , Ligandos , Modelos Moleculares , Movimiento/efectos de los fármacos , Opsinas/agonistas , Opsinas/química , Opsinas/metabolismo , Propanolaminas/química , Propanolaminas/metabolismo , Propanolaminas/farmacología , Conformación Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human ß(2) adrenergic receptor (ß(2)AR) as a guide, we designed a ß(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent ß(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound ß(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 µs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Cristalización , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Agonismo Inverso de Drogas , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Procaterol/química , Procaterol/metabolismo , Propanolaminas/química , Propanolaminas/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ß(2) adrenergic receptor (ß(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ß(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the ß(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ß(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animales , Dominio Catalítico , Bovinos , Cristalización , Cristalografía por Rayos X , Activación Enzimática , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , RatasRESUMEN
The recent, unfortunate death of Alfred G. ("Al") Gilman, M.D., Ph.D., represents a sad signpost for an era spanning over 40 years in molecular pharmacology. Gilman's discoveries, influence, and persona were dominant forces in research and training in pharmacology. Here, we review the progression of ideas and knowledge that spawned early work by Gilman and collaborators (among them, one of the authors) and later efforts (including those of the other author) that have recently yielded a comprehensive and precise structural understanding of fundamental topics in pharmacology: the binding of ligands to G protein-coupled receptors (GPCRs) and the interaction of GPCRs with heterotrimeric G proteins and effector molecules. Those data provide new and important insights into the molecular basis that underlies affinity and efficacy, two of the most important features of drug action, which represent the latest chapter in the saga that Al Gilman's work helped launch.