RESUMEN
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a â¼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
Asunto(s)
Arrestina/química , Arrestina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Humanos , Rayos Láser , Ratones , Modelos Moleculares , Complejos Multiproteicos/biosíntesis , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Transducción de Señal , Rayos XRESUMEN
More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas , Rodopsina , Animales , Bovinos , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica , Rodopsina/química , Rodopsina/genética , Rodopsina/metabolismo , Análisis EspectralRESUMEN
Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of â¼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.
Asunto(s)
Luz , Nanoestructuras/química , Conformación Proteica/efectos de la radiación , Rodopsina/química , Transducina/química , Animales , Bovinos , Estructura Secundaria de Proteína , Rodopsina/metabolismo , Rodopsina/efectos de la radiación , Marcadores de Spin , Transducina/metabolismo , Transducina/efectos de la radiaciónRESUMEN
Electron paramagnetic resonance (EPR) spectroscopy, together with spin labeling techniques, has played a major role in the characterization of rhodopsin, the photoreceptor protein and G protein-coupled receptor (GPCR) in rod cells. Two decades ago, these biophysical tools were the first to identify transmembrane helical movements in rhodopsin upon photo-activation, a critical step in the study of GPCR signaling. EPR methods were employed to identify functional loop dynamics within rhodopsin, to measure light-induced millisecond timescale changes in rhodopsin conformation, to characterize the effects of partial agonists on the apoprotein opsin, and to study lipid interactions with rhodopsin. With the emergence of advanced pulsed EPR techniques, the stage was set to determine the amplitude of structural changes in rhodopsin and the dynamics in the rhodopsin signaling complexes. Work in this area has yielded invaluable information about mechanistic properties of GPCRs. Using EPR techniques, receptors are studied in native-like membrane environments and the effects of lipids on conformational equilibria can be explored. This perspective addresses the impact of EPR methods on rhodopsin and GPCR structural biology, highlighting historical discoveries made with spin labeling techniques, and outlining exciting new directions in the field.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Rodopsina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Rodopsina/químicaRESUMEN
Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications.
Asunto(s)
Canales de Potasio de Rectificación Interna/metabolismo , Ingeniería de Proteínas/métodos , Receptor Muscarínico M2/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acetilcolina/farmacología , Regulación Alostérica , Animales , Dopamina/farmacología , Canales Iónicos Activados por Ligandos/metabolismo , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes de Fusión/metabolismo , XenopusRESUMEN
Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations-F45L, V209M and F220C-yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.
Asunto(s)
Mutación , Mutación Puntual , Retina/metabolismo , Retinitis Pigmentosa/genética , Rodopsina/química , Rodopsina/genética , Animales , Células COS , Bovinos , Chlorocebus aethiops , Proteínas de Unión al GTP/química , Células HEK293 , Humanos , Liposomas/metabolismo , Ratones Noqueados , Proteínas de Transferencia de Fosfolípidos/metabolismo , Multimerización de Proteína , Retina/química , Transducina/genéticaRESUMEN
Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-"uncoupled" receptors such as GPCRs optimized for crystallization by alteration of their third intracellular (i3) loop. One of the most widely used approaches consists in replacing the i3 loop with the T4 phage lysozyme (T4L) domain that obstructs the access of G proteins to their binding site. We recently demonstrated that the ICCR technology can functionally characterize GPCRs(T4L). Two-electrode voltage-clamp (TEVC) recordings revealed that apparent affinities and sensitivities to ligands are not affected by T4L insertion, while ICCRs(T4L) displayed a partial agonist phenotype upon binding of full agonists, suggesting that ICCRs could detect intermediate-active states. This chapter aims to provide exhaustive details from molecular biology steps to electrophysiological recordings for the design and the characterization of ICCRs and ICCRs(T4L).
Asunto(s)
Canales de Potasio de Rectificación Interna/metabolismo , Ingeniería de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Bacteriófago T4/química , Bacteriófago T4/metabolismo , Electrodos , Humanos , Canales Iónicos , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/química , Muramidasa/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodos , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Virales/química , Proteínas Virales/metabolismo , Xenopus laevis/genéticaRESUMEN
Rhodopsin is a class A G protein-coupled receptor (GPCR) that provides important insights into the structure and function of the GPCR superfamily. Bovine rhodopsin is widely used as a model for GPCRs and was the first GPCR whose X-ray crystal structure was solved. One of the advantages of rhodopsin is that it is abundant in native tissue, and as a result, milligram quantities can be purified from the retinal rod cells of bovine eyes. Nonetheless, the study of GPCR conformation and dynamics, e.g., by electron paramagnetic resonance or (19)F nuclear magnetic resonance spectroscopy, typically requires mutagenesis to enable site-directed labeling of the protein. Mutations are also of great importance as they can stabilize the receptor and can be necessary to study different receptor conformations. Recombinant production of rhodopsins for biophysical studies has been achieved in different systems, including mammalian, insect, and yeast cells in culture, and from Drosophila melanogaster and Caenorhabditis elegans tissue. The piggyBac (PB) transposon system is used for gene delivery into a variety of cell types (e.g., HEK293 and CHO cells, fibroblasts, stem cells) and living organisms (e.g., honeybees, pigs, chicken, mice). Recently, the PB transposon has been described as an efficient tool for inducible protein expression in HEK293T and HEK293S N-acetylglucosaminyltransferase I-deficient (GnTI(-)) cells. This chapter describes a protocol for using the PB-based system for inducible expression of bovine rhodopsin in HEK293S GnTI(-) cells. Using this protocol, we expressed and purified 26 rhodopsin mutants to be used for site-directed spin labeling.
Asunto(s)
Clonación Molecular/métodos , Técnicas de Transferencia de Gen , Células HEK293/metabolismo , Rodopsina/genética , Animales , Bovinos , Técnicas de Cultivo de Célula/métodos , Elementos Transponibles de ADN , ADN Complementario/genética , Eliminación de Gen , Humanos , Ratones , N-Acetilglucosaminiltransferasas/genética , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Rodopsina/aislamiento & purificación , Transfección/métodosRESUMEN
Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cisâtrans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.
Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Luz , Canales de Potasio de Rectificación Interna/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Animales , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Oocitos/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Rodopsina/genética , Xenopus laevis/genéticaRESUMEN
Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K(+)-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K(+) channel Kir6.2 and muscarinic M(2) or dopaminergic D(2) receptors. Here, we extend the concept to the distinct, longer ß(2)-adrenergic receptor which, unlike M(2) and D(2) receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of ß(2) constructs. We then demonstrate how engineering of both receptor and channel can produce ß(2)-Kir6.2 ICCRs. Specifically, removal of 62-72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the ß(2) ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the ß(2) receptor.