RESUMEN
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.
Asunto(s)
Rodopsina , Visión Ocular , Animales , Sitios de Unión/efectos de la radiación , Cristalografía , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Isomerismo , Fotones , Unión Proteica/efectos de la radiación , Conformación Proteica/efectos de la radiación , Retinaldehído/química , Retinaldehído/metabolismo , Retinaldehído/efectos de la radiación , Rodopsina/química , Rodopsina/metabolismo , Rodopsina/efectos de la radiación , Factores de Tiempo , Visión Ocular/fisiología , Visión Ocular/efectos de la radiaciónRESUMEN
Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist, 11-cis retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and nonvisual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here, we show that a nonnatural retinal analog, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as a JSR1 agonist. ATR6.11 binding also enables complex formation between JSR1 and signaling partners. Our findings demonstrate the utility of retinal analogs for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.
Asunto(s)
Opsinas , Retinaldehído , Animales , Retinaldehído/metabolismo , Retinaldehído/química , Retinaldehído/análogos & derivados , Opsinas/metabolismo , Opsinas/química , Rodopsina/metabolismo , Rodopsina/química , Arañas/metabolismo , HumanosRESUMEN
Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.
Asunto(s)
Flavobacteriaceae/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efectos de la radiación , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/efectos de la radiación , Sitios de Unión , Cristalografía , Electrones , Transporte Iónico , Isomerismo , Rayos Láser , Protones , Teoría Cuántica , Retinaldehído/química , Retinaldehído/metabolismo , Bases de Schiff/química , Sodio/metabolismo , Análisis Espectral , Electricidad Estática , Factores de TiempoRESUMEN
Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.
Asunto(s)
Calmodulina , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Calmodulina/metabolismo , Activación del Canal Iónico/fisiología , Microscopía por Crioelectrón , Calcio/metabolismo , Nucleótidos Cíclicos/farmacología , GMP Cíclico/metabolismoRESUMEN
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
G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward ß-arrestin-1 and ß-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting ß-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.
Asunto(s)
Prolina/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Sitio Alostérico , Secuencias de Aminoácidos , Clonación Molecular , Dimerización , Células HEK293 , Humanos , Cinética , Ligandos , Péptidos/química , Ingeniería de Proteínas/métodos , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [ß1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using ß1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαißγ and ß-arrestin-1 and showed that carvedilol induces an increase in coupling of ß-arrestin-1 and Gαißγ to ß1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores Opioides/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Arrestina/genética , Arrestina/metabolismo , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ligandos , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores Opioides/química , Anticuerpos de Cadena Única , Pavos , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismoRESUMEN
Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically fixed and stained wild-type mouse retina, including axons and synapses, with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analyzing disease processes like neurodegeneration at sub-200â nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptor synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam/scanning electron microscope tomography. The subcellular structures of synapses obtained with the X-ray technique matched the electron microscopy data, demonstrating that PXCT is a powerful scanning method for tissue volumes of more than 60 cells and sensitive enough for identification of regions as small as 200â nm, which remain available for further structural and biochemical investigations.
Asunto(s)
Retina , Tomografía , Animales , Imagenología Tridimensional , Ratones , Microscopía Electrónica , Sinapsis , Tomografía Computarizada por Rayos XRESUMEN
The recently reported structure of the human CNGA1/CNGB1 CNG channel in the open state (Xue et al., 2021a) shows that one CNGA1 and one CNGB1 subunit do not open the central hydrophobic gate completely upon cGMP binding. This is different from what has been reported for CNGA homomeric channels (Xue et al., 2021b; Zheng et al., 2020). In seeking to understand how this difference is due to the presence of the CNGB1 subunit, we find that the deposited density map (Xue et al., 2021a) (EMDB 24465) contains an additional density not reported in the images of the original publication. This additional density fits well the structure of calmodulin (CaM), and it unambiguously connects the newly identified D-helix of CNGB1 to one of the CNGA1 helices (A1R) participating in the coiled-coil region. Interestingly, the CNGA1 subunit that engages in the interaction with this additional density is the one that, together with CNGB1, does not open completely the central gate. The sequence of the D-helix of CNGB1 contains a known CaM-binding site of exquisitely high affinity - named CaM2 (Weitz et al., 1998) -, and thus the presence of CaM in that region is not surprising. The mechanism through which CaM reduces currents across the membrane by acting on the native channel (Bauer, 1996; Hsu and Molday, 1993; Weitz et al., 1998) remains unclear. We suggest that the presence of CaM may explain the partially open central gate reported by Xue et al. (2021a). The structure of the open and closed states of the CNGA1/CNGB1 channel may be different with and without CaM present.
Asunto(s)
Calmodulina , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Sitios de Unión , Calmodulina/metabolismo , Microscopía por Crioelectrón , Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Humanos , Células Fotorreceptoras Retinianas Bastones/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey ß1-adrenergic receptor (ß1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/metabolismo , Transducción de Señal , Agonistas de Receptores Adrenérgicos beta 1/química , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Antagonistas de Receptores Adrenérgicos beta 1/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Agonismo Parcial de Drogas , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Ligandos , Modelos Moleculares , Movimiento , Mutación Puntual/genética , Estabilidad Proteica , Estructura Secundaria de Proteína/efectos de los fármacos , Receptores Adrenérgicos beta 1/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , PavosRESUMEN
Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses. They are the targets of nearly one-third of all prescribed medicinal drugs such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins. Structural rearrangements of residue contacts in the transmembrane domain serve as 'activation pathways' that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Sitios de Unión , Secuencia Conservada , Humanos , Ligandos , Modelos Moleculares , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/clasificación , Receptores Acoplados a Proteínas G/genética , Receptores de Vasopresinas/química , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Transducción de Señal , Homología Estructural de ProteínaRESUMEN
Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs.
Asunto(s)
Proteínas de Artrópodos/ultraestructura , Rodopsina/ultraestructura , Transducción de Señal/efectos de la radiación , Arañas , Animales , Proteínas de Artrópodos/aislamiento & purificación , Proteínas de Artrópodos/metabolismo , Cristalografía por Rayos X , Células HEK293 , Humanos , Ligandos , Luz , Simulación de Dinámica Molecular , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructura , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Rodopsina/aislamiento & purificación , Rodopsina/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
G-protein coupled receptors (GPCRs) are among the most versatile signal transducers in the cell. Once activated, GPCRs sample a large conformational space and couple to G-proteins to initiate distinct signaling pathways. The dynamical behavior of GPCR-G-protein complexes is difficult characterize structurally, and it might hinder obtaining routine high-resolution density maps in single-particle reconstructions. Here, we used variability analysis on the rhodopsin-Gi-Fab16 complex cryo-EM dataset, and the results provide insights into the dynamic nature of the receptor-complex interaction. We compare the outcome of this analysis with recent results obtained on the cannabinoid-Gi- and secretin-Gs-receptor complexes. Despite differences related to the biochemical compositions of the three samples, a set of consensus movements emerges. We anticipate that systematic variability analysis on GPCR-G-protein complexes may provide useful information not only at the biological level, but also for improving the preparation of more stable samples for cryo-EM single-particle analysis.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Unión al GTP/química , Complejos Multiproteicos/química , Receptores Acoplados a Proteínas G/química , Bases de Datos de Proteínas , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/metabolismo , Imagenología Tridimensional , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Complejos Multiproteicos/metabolismo , Conformación Proteica en Hélice alfa , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/química , Rodopsina/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey ß1-adrenergic receptor (ß1AR) uniformly or selectively labeled in 15N or 2H,15N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2-0.3 mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-ß1AR mutant also comprises the two native tyrosines Y5.58 and Y7.53, which enable G protein binding. High-quality 1H-15N TROSY spectra were obtained for E. coli-expressed YY-ß1AR in three different functional states (antagonist, agonist, and agonist + G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.
Asunto(s)
Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Expresión Génica , Vectores Genéticos/genética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica , Estabilidad Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/aislamiento & purificación , Proteínas RecombinantesRESUMEN
Dynamic hydrogen-bond networks provide proteins with structural plasticity required to translate signals such as ligand binding into a cellular response or to transport ions and larger solutes across membranes and, thus, are of central interest to understand protein reaction mechanisms. Here, we present C-Graphs, an efficient tool with graphical user interface that analyzes data sets of static protein structures or of independent numerical simulations to identify conserved, vs unique, hydrogen bonds and hydrogen-bond networks. For static structures, which may belong to the same protein or to proteins with different sequences, C-Graphs uses a clustering algorithm to identify sites of the hydrogen-bond network where waters are conserved among the structures. Using C-Graphs, we identify an internal protein-water hydrogen-bond network common to static structures of visual rhodopsins and adenosine A2A G protein-coupled receptors (GPCRs). Molecular dynamics simulations of a visual rhodopsin indicate that the conserved hydrogen-bond network from static structure can recruit dynamic hydrogen bonds and extend throughout most of the receptor. We release with this work the code for C-Graphs and its graphical user interface.
Asunto(s)
Rodopsina , Agua , Hidrógeno , Enlace de Hidrógeno , Simulación de Dinámica MolecularRESUMEN
Box jellyfish and vertebrates are separated by >500 million years of evolution yet have structurally analogous lens eyes that employ rhodopsin photopigments for vision. All opsins possess a negatively charged residue-the counterion-to maintain visible-light sensitivity and facilitate photoisomerization of their retinaldehyde chromophore. In vertebrate rhodopsins, the molecular evolution of the counterion position-from a highly conserved distal location in the second extracellular loop (E181) to a proximal location in the third transmembrane helix (E113)-is established as a key driver of higher fidelity photoreception. Here, we use computational biology and heterologous action spectroscopy to determine whether the appearance of the advanced visual apparatus in box jellyfish was also accompanied by changes in the opsin tertiary structure. We found that the counterion in an opsin from the lens eye of the box jellyfish Carybdea rastonii (JellyOp) has also moved to a unique proximal location within the transmembrane bundle-E94 in TM2. Furthermore, we reveal that this Schiff base/counterion system includes an additional positive charge-R186-that has coevolved with E94 to functionally separate E94 and E181 in the chromophore-binding pocket of JellyOp. By engineering this pocket-neutralizing R186 and E94, or swapping E94 with the vertebrate counterion E113-we can recreate versions of the invertebrate and vertebrate counterion systems, respectively, supporting a relatively similar overall architecture in this region of animal opsins. In summary, our data establish the third only counterion site in animal opsins and reveal convergent evolution of tertiary structure in opsins from distantly related species with advanced visual systems.
Asunto(s)
Cubomedusas/genética , Evolución Molecular , Rodopsina , Visión Ocular/genética , Animales , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopsina/química , Rodopsina/genética , Rodopsina/metabolismoRESUMEN
In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.
Asunto(s)
Diseño de Fármacos , Preparaciones Farmacéuticas/administración & dosificación , Conformación Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Receptores Acoplados a Proteínas G/química , Rodopsina/química , Animales , Células Cultivadas , Humanos , Ligandos , Ratones , Modelos Moleculares , Preparaciones Farmacéuticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/metabolismoRESUMEN
Corona virus spike protein S is a large homo-trimeric protein anchored in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, and contribute symmetrically to a relatively large H-bond cluster at the receptor binding domain, and to a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.
Asunto(s)
Betacoronavirus/química , Gráficos por Computador , Infecciones por Coronavirus/virología , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Mapeo de Interacción de Proteínas/métodos , Glicoproteína de la Espiga del Coronavirus , Algoritmos , Enzima Convertidora de Angiotensina 2 , Betacoronavirus/fisiología , COVID-19 , Biología Computacional/métodos , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Pandemias , Peptidil-Dipeptidasa A/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
G protein-coupled receptors (GPCRs) are a large and ubiquitous family of membrane receptors of great pharmacological interest. Cell-based assays are the primary tool for assessing GPCR interactions and activation but their design and intrinsic complexity limit their application. Biosensor-based assays that directly and specifically report GPCR-protein binding (e.g. arrestin or G protein) could provide a good alternative. We present an approach based on the stable immobilization of different arrestin-3 proteins (wild type, and two mutants, mutant X (arrestin-3 I386A) and mutant Y (arrestin-3 R393E)) via histidine tags on NTA(Ni2+)-coated sensors in a defined orientation. Using biolayer interferometry (BLI), surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D), we were able to follow the interaction between the different arrestin-3 proteins and a representative GPCR, jumping spider rhodopsin-1 (JSR1), in a label-free manner in real-time. The interactions were quantified as binding affinity, association and dissociation rate constants. The combination of surface-based biosensing methods indicated that JSR1 showed the strongest binding to arrestin mutant Y. Taken together, this work introduces direct label-free, biosensor-based screening approaches that can be easily adapted for testing interactions of proteins and other compounds with different GPCRs.
Asunto(s)
Proteínas Inmovilizadas/metabolismo , Rodopsina/metabolismo , Arrestina beta 2/metabolismo , Animales , Proteínas de Artrópodos/metabolismo , Técnicas Biosensibles , Proteínas Inmovilizadas/genética , Membrana Dobles de Lípidos/química , Mutación , Fosfatidilcolinas/química , Unión Proteica , Tecnicas de Microbalanza del Cristal de Cuarzo , Arañas/química , Resonancia por Plasmón de Superficie , Arrestina beta 2/genéticaRESUMEN
Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral ß-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open ß-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.