RESUMEN
Lysophospholipids are bioactive lipids and can signal through G-protein-coupled receptors (GPCRs). The best studied lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). The mechanisms of lysophospholipid recognition by an active GPCR, and the activations of lysophospholipid GPCR-G-protein complexes remain unclear. Here we report single-particle cryo-EM structures of human S1P receptor 1 (S1P1) and heterotrimeric Gi complexes formed with bound S1P or the multiple sclerosis (MS) treatment drug Siponimod, as well as human LPA receptor 1 (LPA1) and Gi complexes in the presence of LPA. Our structural and functional data provide insights into how LPA and S1P adopt different conformations to interact with their cognate GPCRs, the selectivity of the homologous lipid GPCRs for S1P versus LPA, and the different activation mechanisms of these GPCRs by LPA and S1P. Our studies also reveal specific optimization strategies to improve the MS-treating S1P1-targeting drugs.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Azetidinas/farmacología , Azetidinas/uso terapéutico , Compuestos de Bencilo/farmacología , Compuestos de Bencilo/uso terapéutico , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/aislamiento & purificación , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Humanos , Lisofosfolípidos/metabolismo , Conformación Molecular/efectos de los fármacos , Simulación del Acoplamiento Molecular , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/aislamiento & purificación , Receptores del Ácido Lisofosfatídico/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Células Sf9 , Imagen Individual de Molécula , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/aislamiento & purificación , Receptores de Esfingosina-1-Fosfato/ultraestructura , SpodopteraRESUMEN
The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.
Asunto(s)
Microscopía por Crioelectrón , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Prurito/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores de Neuropéptido/química , Agonismo Inverso de Drogas , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/ultraestructura , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/ultraestructura , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructura , Receptores de Neuropéptido/metabolismo , Receptores de Neuropéptido/ultraestructuraRESUMEN
Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB1 cannabinoid receptor G-protein coupling in part by altering the selectivity for Gαi subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel ß-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The ß-barrel has a gap between strands ß8 and ß10, which deviates from ß-sandwich fatty acid-binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the ß-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing ß-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gαi N terminus. However, CRIP1a could not bind the nonmyristolyated Gαi peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB1 receptor.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Cannabinoides/metabolismo , Proteínas Portadoras/genética , Endocannabinoides , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB1/ultraestructura , Receptores de Cannabinoides/metabolismo , Receptores de Cannabinoides/ultraestructuraAsunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Lípidos/química , Conformación Proteica , Receptores de Neurotensina/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Receptores de Neurotensina/química , Receptores de Neurotensina/genéticaRESUMEN
G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP Heterotriméricas/ultraestructura , Membrana Dobles de Lípidos , Nanoestructuras/química , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/ultraestructura , Regulación Alostérica , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/ultraestructura , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/ultraestructura , Guanosina Difosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Micelas , Modelos Moleculares , Neurotensina/química , Neurotensina/metabolismo , Conformación Proteica , Receptores de Neurotensina/química , Transducción de SeñalRESUMEN
Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.
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/metabolismo , Glucocorticoides/química , Glucocorticoides/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/ultraestructura , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Humanos , Ligandos , Lipoilación , Modelos Moleculares , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
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
Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1 These two structures adopt a similar open binding cavity to accommodate Gs and Gi1 The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Glucagón/química , Receptores de Glucagón/química , Sitios de Unión , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Glucagón/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Conformación Proteica en Hélice alfa , Receptores de Glucagón/metabolismo , Receptores de Glucagón/ultraestructura , Transducción de SeñalRESUMEN
Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.
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 , Receptores de Neurotensina/química , Receptores de Neurotensina/ultraestructura , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Oligopéptidos/química , Oligopéptidos/farmacología , Unión Proteica , Conformación Proteica , Receptores de Neurotensina/agonistas , Receptores de Neurotensina/metabolismoRESUMEN
The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins3. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.
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 , Oxiesteroles/química , Receptor Smoothened/química , Receptor Smoothened/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Ligandos , Modelos Moleculares , Oxiesteroles/metabolismo , Receptor Patched-1/metabolismo , Conformación Proteica , Transducción de Señal , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/químicaRESUMEN
G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein ßγ subunit (Gßγ), which is released from the heterotrimeric G protein (Gαßγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oßγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oßγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.
Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades beta de la Proteína de Unión al GTP/ultraestructura , Subunidades gamma de la Proteína de Unión al GTP/ultraestructura , Activación del Canal Iónico/fisiología , Transferencia de Energía por Resonancia de Bioluminiscencia , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/aislamiento & purificación , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades beta de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
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
G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of Gs to four different GPCRs have been elucidated1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT1B receptor (5-HT1BR) bound to the agonist donitriptan and coupled to an engineered Go heterotrimer. In this complex, 5-HT1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the ß2-adrenoceptor (ß2AR) 3 or the adenosine A2A receptor (A2AR) 1 in complex with Gs. In contrast to the complexes with Gs, the gap between the receptor and the Gß-subunit in the Go-5-HT1BR complex precludes molecular contacts, and the interface between the Gα-subunit of Go and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the Go α-subunit. The molecular variations between the interfaces of Go and Gs in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Receptor de Serotonina 5-HT1B/metabolismo , Receptor de Serotonina 5-HT1B/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , 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 , Nitrilos/química , Nitrilos/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Conformación Proteica , Receptor de Serotonina 5-HT1B/química , Agonistas del Receptor de Serotonina 5-HT1/química , Agonistas del Receptor de Serotonina 5-HT1/metabolismo , Triptaminas/química , Triptaminas/metabolismoRESUMEN
G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, Gi-bound and arrestin-bound forms of rhodopsin with inactive and Gs-bound forms of the ß2-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of Gs, Gi and arrestin by activated G-protein-coupled receptors.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Rodopsina/metabolismo , Rodopsina/ultraestructura , Arrestina/química , Arrestina/metabolismo , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , 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 , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Rodopsina/química , Transducción de Señal , Especificidad por SustratoRESUMEN
The µ-opioid receptor (µOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by µOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the µOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the µOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR.
Asunto(s)
Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Receptores Opioides mu/metabolismo , Receptores Opioides mu/ultraestructura , Animales , Sitios de Unión , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Ligandos , Ratones , Ratones Endogámicos BALB C , Simulación de Dinámica Molecular , Morfinanos/química , Morfinanos/metabolismo , Estabilidad Proteica/efectos de los fármacos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/química , Especificidad por SustratoRESUMEN
The heterotrimeric G proteins (Gαßγ) act as molecular switches to mediate signal transduction from G protein-coupled receptors to downstream effectors. Upon interaction with an activated receptor, G protein exchanges its bound GDP with GTP, stimulating downstream signal transmission. Release of GDP requires a structural rearrangement between the GTPase domain and helical domain of the Gα subunit. Here, we used single molecule fluorescence resonance energy transfer (smFRET) technique to study the conformational dynamics of these two domains in the apo state and in the binding of different ligands. Direct imaging of individual molecules showed that the Giα subunit is highly dynamic, and at least three major conformations of Giα could be observed in the apo state. Upon binding of GDP, Giα becomes dramatically less dynamic, resulting in a closed conformation between the two domains. We postulate that changes between the three conformations are sequential, and the three conformations appear to have distinct affinities toward GDP.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Guanosina Difosfato/química , Imagen Molecular/métodos , Sitios de Unión , Activación Enzimática , Unión Proteica , Conformación Proteica , Dominios Proteicos , Subunidades de ProteínaRESUMEN
A method is introduced to construct a better approximation for the reaction coordinate for protein folding from known order parameters. The folding of a two-state off-lattice alpha helical Go-type protein is studied using molecular dynamics simulations. Folding times are computed directly from simulation, as well as theoretically using an equation derived by considering Brownian-type dynamics for the putative reaction coordinate. Theoretical estimates of the folding time using the number of native contacts (Qn) as a reaction coordinate were seen to differ quite significantly from the true folding time of the protein. By considering the properties of the bimodal free energy surface of this protein as a function of Qn and another relevant coordinate for folding Q (the total number of contacts), we show that by introducing a rotation in the phase space of the order parameters Q and Qn, we can construct a new reaction coordinate q that leads to a fivefold improvement in the estimate of the folding rate. This new coordinate q, resulting from the rotation, lies along the line connecting the unfolded and folded ensemble minima of the free energy map plotted as a function of the original order parameters Q and Qn. Possible reasons for the remaining discrepancy between the folding time computed theoretically and from folding simulations are discussed.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Modelos Químicos , Modelos Moleculares , Simulación por Computador , Cinética , Transición de Fase , Conformación Proteica , Pliegue de Proteína , RotaciónRESUMEN
In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism.