RESUMEN
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Asunto(s)
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Activación del Canal Iónico , Animales , Channelrhodopsins/ultraestructura , Microscopía por Crioelectrón , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , Optogenética , Filogenia , Ratas Sprague-Dawley , Bases de Schiff/química , Células Sf9 , Relación Estructura-ActividadRESUMEN
Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.
Asunto(s)
Bacteriorodopsinas/ultraestructura , Rayos Láser , Protones , Retinaldehído/química , Difracción de Rayos X/métodos , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Cristalografía/instrumentación , Cristalografía/métodos , Halobacterium salinarum/química , Halobacterium salinarum/metabolismo , Transporte Iónico , Modelos Moleculares , Conformación Proteica , Retinaldehído/metabolismo , Sincrotrones/instrumentación , Rayos XRESUMEN
Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.
Asunto(s)
Oxígeno , Complejo de Proteína del Fotosistema II , Biocatálisis/efectos de la radiación , Calcio/metabolismo , Cristalografía , Transporte de Electrón/efectos de la radiación , Electrones , Manganeso/metabolismo , Oxidación-Reducción/efectos de la radiación , Oxígeno/química , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema II/efectos de la radiación , Protones , Factores de Tiempo , Tirosina/metabolismo , Agua/química , Agua/metabolismoRESUMEN
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
Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.
Asunto(s)
Microscopía por Crioelectrón , Virus de la Hepatitis B , Transportadores de Anión Orgánico Sodio-Dependiente , Receptores Virales , Simportadores , Animales , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Bovinos , Virus de la Hepatitis B/metabolismo , Hepatocitos/metabolismo , Humanos , Mutación , Transportadores de Anión Orgánico Sodio-Dependiente/química , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/ultraestructura , Ratas , Receptores Virales/química , Receptores Virales/genética , Receptores Virales/metabolismo , Receptores Virales/ultraestructura , Sodio/metabolismo , Simportadores/química , Simportadores/genética , Simportadores/metabolismo , Simportadores/ultraestructuraRESUMEN
Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.
Asunto(s)
Ácidos y Sales Biliares , Microscopía por Crioelectrón , Virus de la Hepatitis B , Transportadores de Anión Orgánico Sodio-Dependiente , Receptores Virales , Simportadores , Anticuerpos , Ácidos y Sales Biliares/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatocitos/metabolismo , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/química , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/ultraestructura , Receptores Virales/química , Receptores Virales/metabolismo , Receptores Virales/ultraestructura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestructuraRESUMEN
Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.
Asunto(s)
Canales de Cloruro/química , Rayos Láser , Canales de Cloruro/metabolismo , Cristalografía , Citoplasma/metabolismo , Transporte Iónico , Luz , Conformación Proteica , Rayos XRESUMEN
Rational synthetic expansion of photoresponsive ligands is important for photopharmacological studies. Adenosine A2A receptor (A2AR) is stimulated by adenosine and related in Parkinson's disease and other diseases. Here, we report the crystal structure of the A2AR in complex with the novel photoresponsive ligand photoNECA (blue) at 3.34 Å resolution. PhotoNECA (blue) was designed for this structural study and the cell-based assay showed a photoresponsive and receptor selective characteristics of photoNECA (blue) for A2AR. The crystal structure explains the binding mode, photoresponsive mechanism and receptor selectivity of photoNECA (blue). Our study would promote not only the rational design of photoresponsive ligands but also dynamic structural studies of A2AR.
Asunto(s)
Receptor de Adenosina A2A , Humanos , Adenosina/metabolismo , Ligandos , Enfermedad de Parkinson , Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/metabolismo , Fotoquímica/métodos , Colorantes Fluorescentes/químicaRESUMEN
MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.
Asunto(s)
Antiportadores/química , Proteínas Bacterianas/química , Activación del Canal Iónico/fisiología , Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión/efectos de los fármacos , Transporte Biológico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Citoplasma/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Cinética , Magnesio/metabolismo , Magnesio/farmacología , Modelos Moleculares , Dominios Proteicos/efectos de los fármacos , Dominios Proteicos/fisiología , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Thermus thermophilus/metabolismoRESUMEN
KW-6356 is a novel adenosine A2A (A2A) receptor antagonist/inverse agonist, and its efficacy as monotherapy in Parkinson's disease (PD) patients has been reported. Istradefylline is a first-generation A2A receptor antagonist approved for use as adjunct treatment to levodopa/decarboxylase inhibitor in adult PD patients experiencing "OFF" episodes. In this study, we investigated the in vitro pharmacological profile of KW-6356 as an A2A receptor antagonist/inverse agonist and the mode of antagonism and compared them with istradefylline. In addition, we determined cocrystal structures of A2A receptor in complex with KW-6356 and istradefylline to explore the structural basis of the antagonistic properties of KW-6356. Pharmacological studies have shown that KW-6356 is a potent and selective ligand for the A2A receptor (the -log of inhibition constant = 9.93 ± 0.01 for human receptor) with a very low dissociation rate from the receptor (the dissociation kinetic rate constant = 0.016 ± 0.006 minute-1 for human receptor). In particular, in vitro functional studies indicated that KW-6356 exhibits insurmountable antagonism and inverse agonism, whereas istradefylline exhibits surmountable antagonism. Crystallography of KW-6356- and istradefylline-bound A2A receptor have indicated that interactions with His2506.52 and Trp2466.48 are essential for the inverse agonism, whereas the interactions at both deep inside the orthosteric pocket and the pocket lid stabilizing the extracellular loop conformation may contribute to the insurmountable antagonism of KW-6356. These profiles may reflect important differences in vivo and help predict better clinical performance. SIGNIFICANCE STATEMENT: KW-6356 is a potent and selective adenosine A2A receptor antagonist/inverse agonist and exhibits insurmountable antagonism, whereas istradefylline, a first-generation adenosine A2A receptor antagonist, exhibits surmountable antagonism. Structural studies of adenosine A2A receptor in complex with KW-6356 and istradefylline explain the characteristic differences in the pharmacological properties of KW-6356 and istradefylline.
Asunto(s)
Antagonistas del Receptor de Adenosina A2 , Agonismo Inverso de Drogas , Enfermedad de Parkinson , Receptor de Adenosina A2A , Humanos , Antagonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Levodopa/farmacología , Levodopa/uso terapéutico , Receptor de Adenosina A2A/fisiologíaRESUMEN
Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ß-barrel across the time window of our measurements.
Asunto(s)
Colorantes , Proteínas , Cristalografía , Estructura Secundaria de ProteínaRESUMEN
Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique µ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.
Asunto(s)
Cristalografía/métodos , Electrones , Rayos Láser , Luz , Oxígeno/química , Oxígeno/efectos de la radiación , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/efectos de la radiación , Biocatálisis/efectos de la radiación , Cianobacterias/química , Transporte de Electrón/efectos de la radiación , Análisis de Fourier , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/metabolismo , Proteínas de Hierro no Heme/efectos de la radiación , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Temperatura , Factores de Tiempo , Agua/química , Agua/metabolismoRESUMEN
Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo.
Asunto(s)
Flavoproteínas/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Optogenética , Fotones , Animales , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , RatonesRESUMEN
Prostanoids are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G-protein-coupled receptors (GPCRs). Here, we report the crystal structure of human prostaglandin (PG) E receptor subtype EP3 bound to endogenous ligand PGE2 at 2.90 Å resolution. The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands.
Asunto(s)
Subtipo EP3 de Receptores de Prostaglandina E/agonistas , Subtipo EP3 de Receptores de Prostaglandina E/química , Sitios de Unión , Cristalografía por Rayos X , Dinoprostona/química , Dinoprostona/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Subtipo EP3 de Receptores de Prostaglandina E/genética , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.
Asunto(s)
Subtipo EP4 de Receptores de Prostaglandina E/química , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Regulación Alostérica , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Caprilatos/química , Caprilatos/metabolismo , Cristalografía por Rayos X , Epoprostenol/análogos & derivados , Epoprostenol/química , Epoprostenol/metabolismo , Humanos , Ligandos , Membrana Dobles de Lípidos , Simulación del Acoplamiento Molecular , Naftalenos/química , Naftalenos/metabolismo , Éteres Fenílicos/química , Éteres Fenílicos/metabolismo , Fenilbutiratos/química , Fenilbutiratos/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/genética , Spodoptera/genéticaRESUMEN
The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.
Asunto(s)
Fructosa/metabolismo , Transportador de Glucosa de Tipo 5/química , Transportador de Glucosa de Tipo 5/metabolismo , Animales , Sitios de Unión , Transporte Biológico , Bovinos , Membrana Celular/metabolismo , Cristalografía por Rayos X , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fructosa/química , Glucosa/química , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/química , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 5/genética , Modelos Moleculares , Mutación Puntual/genética , Conformación Proteica , Ratas , Sales (Química)/química , Electricidad Estática , Relación Estructura-Actividad , Especificidad por Sustrato/genética , Simportadores/química , Simportadores/metabolismoRESUMEN
Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5' AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4 Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure-function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes.
Asunto(s)
Receptores de Adiponectina/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Histidina/química , Histidina/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Adiponectina/metabolismo , Relación Estructura-Actividad , Zinc/metabolismoRESUMEN
Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.
Asunto(s)
Antagonistas Muscarínicos/metabolismo , Pirenzepina/análogos & derivados , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Estabilidad de Enzimas , Humanos , Simulación de Dinámica Molecular , Antagonistas Muscarínicos/química , Mutación , N-Metilescopolamina/química , N-Metilescopolamina/metabolismo , Pirenzepina/química , Pirenzepina/metabolismo , Receptor Muscarínico M2/antagonistas & inhibidoresRESUMEN
The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.
Asunto(s)
Protones , Proteínas de la Matriz Viral/química , Secuencias de Aminoácidos , Enlace de Hidrógeno , Activación del Canal Iónico , Simulación de Dinámica Molecular , Dominios Proteicos , Electricidad Estática , Temperatura , Proteínas de la Matriz Viral/metabolismoRESUMEN
CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The â¼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located â¼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.