RESUMEN
G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular ß sheet with the N-terminal ß strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to ß-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.
Asunto(s)
Arrestinas/química , Rodopsina/química , Secuencia de Aminoácidos , Animales , Arrestinas/metabolismo , Cromatografía Liquida , Humanos , Ratones , Modelos Moleculares , Fosforilación , Ratas , Rodopsina/metabolismo , Alineación de Secuencia , Espectrometría de Masas en Tándem , Rayos XRESUMEN
Cardiac disease remains the leading cause of morbidity and mortality worldwide. The ß1-adrenergic receptor (ß1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by ß1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which ß1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the ß6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Isoproterenol/metabolismo , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/metabolismo , Animales , Sitios de Unión , Bovinos , Línea Celular , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.
Asunto(s)
Fosfolípidos , Receptores Acoplados a Proteínas G , Animales , Transporte Biológico , Colesterol , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Bovinos , PavosRESUMEN
The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.
Asunto(s)
Antivirales , COVID-19 , Animales , Humanos , SARS-CoV-2 , Pandemias , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Epítopos , Glicoproteína de la Espiga del CoronavirusRESUMEN
Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.
Asunto(s)
Glucagón/análogos & derivados , Receptores de Glucagón/química , Receptores de Glucagón/metabolismo , Cristalografía por Rayos X , Agonismo Parcial de Drogas , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
In the PDF version of this Article, owing to a typesetting error, an incorrect figure was used for Extended Data Fig. 5; the correct figure was used in the HTML version. This has been corrected online.
RESUMEN
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 ubiquitin-proteasome system fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adapter proteins Sts1 and Blm10, while in mammalian cells, postmitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adapter protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, that is quiescence, are associated with a decline in ATP and the reorganization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in an ubiquitin-dependent manner into motile and reversible proteasome storage granules in the cytoplasm. In cancer cells, upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing polyubiquitinated substrates are formed by liquid-liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling, and the intracellular organization of proteasomes during cellular stress conditions.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Saccharomyces cerevisiae , Transporte Activo de Núcleo Celular , Adenosina Trifosfato/metabolismo , Animales , Citoplasma/metabolismo , Mamíferos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the ß2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease.
Asunto(s)
Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/metabolismo , Agonistas del Receptor de Adenosina A2/farmacología , Regulación Alostérica/efectos de los fármacos , Agonismo Inverso de Drogas , Agonismo Parcial de Drogas , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Modelos Biológicos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica/efectos de los fármacos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , TermodinámicaRESUMEN
Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.
Asunto(s)
Cianobacterias/química , Mutación , Bombas de Protones/química , Rodopsinas Microbianas/química , Sitios de Unión , Biopolímeros/química , Cristalografía por Rayos X , Transporte Iónico , Conformación Proteica , Bombas de Protones/genética , Protones , Retinaldehído/metabolismo , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismoRESUMEN
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
Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.
Asunto(s)
Nanopartículas/química , Multimerización de Proteína , Rodopsina/química , Animales , Bovinos , Microscopía por Crioelectrón , Células HEK293 , Humanos , Dominios Proteicos , Rodopsina/ultraestructuraRESUMEN
Site-specific modification of proteins with functional molecules provides powerful tools for researching and engineering proteins. Here we report a new chemical conjugation method which photocages highly reactive but chemically selective moieties, enabling the use of protein-inert amines for selective protein modification. New amino acids FnbY and FmnbY, bearing photocaged quinone methides (QMs), were genetically incorporated into proteins. Upon light activation, they generated highly reactive QM, which rapidly reacted with amine derivatives. This method features a rare combination of desired properties including fast kinetics, small and stable linkage, compatibility with low temperature, photocontrollability, and widely available reagents. Moreover, labeling via FnbY occurs on the ß-carbon, affording the shortest linkage to protein backbone which is essential for advanced studies involving orientation and distance. We installed various functionalities onto proteins and attached a spin label as close as possible to the protein backbone, achieving high resolution in double electron-electron paramagnetic resonance distance measurements.
Asunto(s)
Aminas/química , Indolquinonas/química , Proteínas/química , Coloración y Etiquetado/métodos , Aminoácidos/química , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Procesos Fotoquímicos , Conformación Proteica , Procesamiento Proteico-Postraduccional , Solventes/química , Marcadores de Spin , Compuestos de Sulfhidrilo/química , TemperaturaRESUMEN
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
Protein production remains a major bottleneck in membrane protein structural biology. In many cases, large-scale recombinant protein expression is either unfeasible or impossible, driving structural biologists to explore new production avenues. Several membrane proteins have been successfully refolded from solubilized E. coli inclusion bodies. In recent years, a structure of the G-protein-coupled receptor CXCR1 was obtained using refolded material from E. coli inclusion bodies. However, aggregation during the refolding process is a common difficulty, which is often addressed by immobilization of the protein onto a solid support. Most spectroscopic methods are incompatible with these light-scattering matrices, which renders automated buffer exchange to screen refolding conditions impossible. This work explores a potential approach to overcome this problem by utilizing site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of protein bound to standard, commercially available Ni-NTA agarose resin. With this approach, the correct protein fold is determined by activity, which is inferred from a protein conformational response to a known stimulant. EPR spectra at each state of the refolding workflow of spin-labeled Haloarcula marismortui bacteriorhodopsin-I (HmbRI) are obtained, and refolded fractions of HmbRI with this platform are quantitated using both protein from inclusion bodies and denatured recombinant protein from E. coli membranes. The stimulant used for HmbRI is visible light. The solid support allows for multiple refolding trials through buffer exchanges, and the EPR spectra are collected on the order of seconds under ambient conditions.
Asunto(s)
Bacteriorodopsinas/análisis , Haloarcula marismortui/química , Proteínas de la Membrana/análisis , Espectroscopía de Resonancia por Spin del Electrón , Conformación Proteica , Replegamiento Proteico , Proteínas Recombinantes/análisisRESUMEN
Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins and initiate G-protein-independent signalling. Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs. Structures of the inactive state of arrestin are available, but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a â¼21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor-binding loops in the central crest region include the finger loop, loop 139 (refs 8, 10, 11) and the sequence Asp 296-Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system.
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Arrestinas/química , Arrestinas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Empalme Alternativo , Animales , Arrestinas/genética , Bovinos , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Peso Molecular , Conformación Proteica , Isoformas de Proteínas/genética , Rotación , Eliminación de Secuencia , Electricidad Estática , beta-ArrestinasRESUMEN
Retinal-binding proteins, mainly known as rhodopsins, function as photosensors and ion transporters in a wide range of organisms. From halobacterial light-driven proton pump, bacteriorhodopsin, to bovine photoreceptor, visual rhodopsin, they have served as prototypical α-helical membrane proteins in a large number of biophysical studies and aided in the development of many cutting-edge techniques of structural biology and biospectroscopy. In the last decade, microbial and animal rhodopsin families have expanded significantly, bringing into play a number of new interesting structures and functions. In this review, we will discuss recent advances in biophysical approaches to retinal-binding proteins, primarily microbial rhodopsins, including those in optical spectroscopy, X-ray crystallography, nuclear magnetic resonance, and electron paramagnetic resonance, as applied to such fundamental biological aspects as protein oligomerization, folding, and structure.
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Bacteriorodopsinas/química , Pliegue de Proteína , Multimerización de Proteína , Rodopsina/química , Animales , Bacteriorodopsinas/metabolismo , Bovinos , Cristalografía por Rayos X/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Estructura Cuaternaria de Proteína , Rodopsina/metabolismoRESUMEN
Calmodulin is a ubiquitous calcium sensor protein, known to serve as a critical interaction hub with a wide range of signaling partners. While the holo form of calmodulin (CaM-4Ca2+) has a well-defined ground state structure, it has been shown to undergo exchange, on a millisecond timescale, to a conformation resembling that of the peptide bound state. Tagged paramagnetic relaxation agents have been previously used to identify long-range dipolar interactions through relaxation effects on nuclear spins of interest. In the case of calmodulin, this lead to the determination of the relative orientation of the N- and C-terminal domains and the presence of a weakly populated peptide bound like state. Here, we make use of pseudocontact shifts from a tagged paramagnetic shift reagent which allows us to define minor states both in 13C and 15N NMR spectra and through 13C- and 15N-edited 1H-CPMG relaxation dispersion measurements. This is validated by pulsed EPR (DEER) spectroscopy which reveals an ensemble consisting of a compact peptide-bound like conformer, an intermediate peptide-bound like conformer, and a (dumbbell-like) extended ground state conformer of CaM-4Ca2+, where addition of the MLCK peptide increases the population of the peptide-bound conformers. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.
Asunto(s)
Calmodulina/química , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Humanos , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαßγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 Å and 2.85 Å crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.
Asunto(s)
Rodopsina/química , Rodopsina/metabolismo , Sitios de Unión , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Ligandos , Modelos Moleculares , Opsinas/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Retinaldehído/química , Retinaldehído/metabolismo , Bases de Schiff/química , Electricidad EstáticaRESUMEN
Pulsed electron paramagnetic resonance experiments can measure individual distances between two spin-labeled side chains in proteins in the range of â¼1.5-8 nm. However, the flexibility of traditional spin-labeled side chains leads to diffuse spin density loci and thus distance distributions with relatively broad peaks, thereby complicating the interpretation of protein conformational states. Here we analyzed the spin-labeled V1 side chain, which is internally anchored and hence less flexible. Crystal structures of V1-labeled T4 lysozyme constructs carrying the V1 side chain on α-helical segments suggest that V1 side chains adopt only a few discrete rotamers. In most cases, only one rotamer is observed at a given site, explaining the frequently observed narrow distance distribution for doubly V1-labeled proteins. We used the present data to derive guidelines that may allow distance interpretation of other V1-labeled proteins for higher-precision structural modeling.