RESUMO
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.
Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Anticorpos , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Receptores Virais/química , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestruturaRESUMO
Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hß2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.
Assuntos
Galanina/química , Proteínas Heterotriméricas de Ligação ao GTP , Receptor Tipo 2 de Galanina/química , Microscopia Crioeletrônica , Galanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Receptor Tipo 2 de Galanina/metabolismoRESUMO
Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.
Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Rodopsinas Microbianas/metabolismo , Cátions Monovalentes/metabolismo , Canais de Cloreto/isolamento & purificação , Canais de Cloreto/efeitos da radiação , Canais de Cloreto/ultraestrutura , Cristalografia/métodos , Radiação Eletromagnética , Lasers , Simulação de Dinâmica Molecular , Nocardioides , Conformação Proteica em alfa-Hélice/efeitos da radiação , Estrutura Terciária de Proteína/efeitos da radiação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Proteínas Recombinantes/ultraestrutura , Retinaldeído/metabolismo , Retinaldeído/efeitos da radiação , Rodopsinas Microbianas/isolamento & purificação , Rodopsinas Microbianas/efeitos da radiação , Rodopsinas Microbianas/ultraestrutura , Água/metabolismoRESUMO
Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial. Here we captured the CO migration processes in the tense (T), relaxed (R), and second relaxed (R2) quaternary structures of human hemoglobin by crystallography using a high-repetition pulsed laser technique at cryogenic temperatures. We found that in each quaternary structure, the photodissociated CO molecules migrate along distinct pathways in the α and ß subunits by hopping between the internal cavities with correlated side chain motions of large nonpolar residues, such as α14Trp(A12), α105Leu(G12), ß15Trp(A12), and ß71Phe(E15). We also observe electron density evidence for the distal histidine [α58/ß63His(E7)] swing-out motion regardless of the quaternary structure, although less evident in α subunits than in ß subunits, suggesting that some CO molecules have escaped directly through the E7 gate. Remarkably, in T-state Fe(II)-Ni(II) hybrid hemoglobins in which either the α or ß subunits contain Ni(II) heme that cannot bind CO, the photodissociated CO molecules not only dock at the cavities in the original Fe(II) subunit, but also escape from the protein matrix and enter the cavities in the adjacent Ni(II) subunit even at 95 K, demonstrating the high gas permeability and porosity of the hemoglobin molecule. Our results provide a comprehensive picture of ligand movements in hemoglobin and highlight the relevance of cavities, nonpolar residues, and distal histidines in facilitating the ligand migration.
Assuntos
Hemoglobinas/química , Hemoglobinas/metabolismo , Monóxido de Carbono/metabolismo , Cristalografia por Raios X , Difusão , Heme/química , Histidina/química , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de FusãoRESUMO
Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or ß subunit contains a ferric heme in the α2ß2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His â tyrosine [Tyr]) and M Boston (α58His â Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.
Assuntos
Hemoglobina M , Cristalografia , Teoria da Densidade Funcional , Heme/química , Hemoglobina M/química , Hemoglobina M/metabolismo , Humanos , Análise Espectral Raman , Tirosina/química , VibraçãoRESUMO
Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and noncompetitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, coimmunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affects the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. IMPORTANCE Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.
Assuntos
Vírus da Hepatite B/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Multimerização Proteica , Receptores Virais/metabolismo , Simportadores/metabolismo , Internalização do Vírus/efeitos dos fármacos , Transporte Biológico , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Troglitazona/farmacologia , Ligação Viral/efeitos dos fármacosRESUMO
Sodium taurocholate cotransporting polypeptide (NTCP) is a host cell receptor required for hepatitis B virus (HBV) entry. However, the susceptibility of NTCP-expressing cells to HBV is diverse depending on the culture condition. Stimulation with epidermal growth factor (EGF) was found to potentiate cell susceptibility to HBV infection. Here, we show that EGF receptor (EGFR) plays a critical role in HBV virion internalization. In EGFR-knockdown cells, HBV or its preS1-specific fluorescence peptide attached to the cell surface, but its internalization was attenuated. PreS1 internalization and HBV infection could be rescued by complementation with functional EGFR. Interestingly, the HBV/preS1-NTCP complex at the cell surface was internalized concomitant with the endocytotic relocalization of EGFR. Molecular interaction between NTCP and EGFR was documented by immunoprecipitation assay. Upon dissociation from functional EGFR, NTCP no longer functioned to support viral infection, as demonstrated by either (i) the introduction of NTCP point mutation that disrupted its interaction with EGFR, (ii) the detrimental effect of decoy peptide interrupting the NTCP-EGFR interaction, or (iii) the pharmacological inactivation of EGFR. Together, these data support the crucial role of EGFR in mediating HBV-NTCP internalization into susceptible cells. EGFR thus provides a yet unidentified missing link from the cell-surface HBV-NTCP attachment to the viral invasion beyond the host cell membrane.
Assuntos
Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Simportadores , Internalização do Vírus , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células Hep G2 , Vírus da Hepatite B/patogenicidade , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMO
Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4ß (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.
Assuntos
Endocitose/genética , Endossomos/genética , Receptores ErbB/genética , Hepatite B/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Receptores ErbB/química , Células Hep G2 , Hepacivirus/química , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , MAP Quinase Quinase 1/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio , Fosfatidilinositol 3-Quinases/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fatores de Transcrição STAT/genética , Simportadores , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Internalização do VírusRESUMO
Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.
Assuntos
Actinomycetales/química , Canais de Cloreto/química , Rodopsinas Microbianas/química , Cristalografia por Raios X , Domínios ProteicosRESUMO
The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.
Assuntos
Adenilil Ciclases/metabolismo , Adenilil Ciclases/fisiologia , Adenilil Ciclases/genética , Sítio Alostérico , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Cianobactérias/metabolismo , AMP Cíclico/metabolismo , Flavinas/metabolismo , Humanos , Luz , Optogenética/métodos , Oscillatoria/metabolismo , Domínios Proteicos , Estrutura Terciária de ProteínaRESUMO
Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.
Assuntos
Adenilil Ciclases/química , Proteínas de Bactérias/química , AMP Cíclico/química , Oscillatoria/enzimologia , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , Ativação Enzimática/genética , Ativação Enzimática/efeitos da radiação , Células HEK293 , Humanos , Luz , Oscillatoria/genética , Domínios Proteicos , Sistemas do Segundo Mensageiro/genética , Sistemas do Segundo Mensageiro/efeitos da radiação , Relação Estrutura-AtividadeRESUMO
Although X-ray crystallography is the most commonly used technique for studying the molecular structure of proteins, it is not generally able to monitor the dynamic changes or global domain motions that often underlie allostery. These motions often prevent crystal growth or reduce crystal order. We have recently discovered a crystal form of human hemoglobin that contains three protein molecules allowed to express a full range of quaternary structures, whereas maintaining strong X-ray diffraction. Here we use this crystal form to investigate the effects of two allosteric effectors, phosphate and bezafibrate, by tracking the structures and functions of the three hemoglobin molecules following the addition of each effector. The X-ray analysis shows that the addition of either phosphate or bezafibrate not only induces conformational changes in a direction from a relaxed-state to a tense-state, but also within relaxed-state populations. The microspectrophotometric O2 equilibrium measurements on the crystals demonstrate that the binding of each effector energetically stabilizes the lowest affinity conformer more strongly than the intermediate affinity one, thereby reducing the O2 affinity of tense-state populations, and that the addition of bezafibrate causes an â¼5-fold decrease in the O2 affinity of relaxed-state populations. These results show that the allosteric pathway of hemoglobin involves shifts of populations rather than a unidirectional conversion of one quaternary structure to another, and that minor conformers of hemoglobin may have a disproportionate effect on the overall O2 affinity.
Assuntos
Modelos Moleculares , Oxigênio/metabolismo , alfa-Globinas/metabolismo , Globinas beta/metabolismo , Algoritmos , Regulação Alostérica , Bezafibrato/química , Bezafibrato/metabolismo , Cristalografia por Raios X , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Cinética , Ligantes , Oxirredução , Oxigênio/química , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Redobramento de Proteína , Estabilidade Proteica , Estrutura Quaternária de Proteína , alfa-Globinas/química , Globinas beta/químicaRESUMO
Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.
Assuntos
Antivirais/farmacologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Células Hep G2 , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/patogenicidade , Hepatite D/tratamento farmacológico , Humanos , Terapia de Alvo Molecular/métodos , Precursores de Proteínas/metabolismo , Sirolimo/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
Hydroxycarboxylic acid receptors (HCAR1, HCAR2, and HCAR3) transduce Gi/o signaling upon biding to molecules such as lactic acid, butyric acid and 3-hydroxyoctanoic acid, which are associated with lipolytic and atherogenic activity, and neuroinflammation. Although many reports have elucidated the function of HCAR2 and its potential as a therapeutic target for treating not only dyslipidemia but also neuroimmune disorders such as multiple sclerosis and Parkinson's disease, the structural basis of ligand recognition and ligand-induced Gi-coupling remains unclear. Here we report three cryo-EM structures of the human HCAR2-Gi signaling complex, each bound with different ligands: niacin, acipimox or GSK256073. All three agonists are held in a deep pocket lined by residues that are not conserved in HCAR1 and HCAR3. A distinct hairpin loop at the HCAR2 N-terminus and extra-cellular loop 2 (ECL2) completely enclose the ligand. These structures also reveal the agonist-induced conformational changes propagated to the G-protein-coupling interface during activation. Collectively, the structures presented here are expected to help in the design of ligands specific for HCAR2, leading to new drugs for the treatment of various diseases such as dyslipidemia and inflammation.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Ácidos Carboxílicos , Ligantes , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Understanding the structure and functional mechanisms of cyanobacterial halorhodopsin has become increasingly important, given the report that Synechocystis halorhodopsin (SyHR), a homolog of the cyanobacterial halorhodopsin from Mastigocladopsis repens (MrHR), can take up divalent ions, such as SO42-, as well as chloride ions. Here, the crystal structure of MrHR, containing a unique "TSD" chloride ion conduction motif, was determined as a homotrimer at a resolution of 1.9â¯Å. The detailed structure of MrHR revealed a unique trimeric topology of the light-driven chloride pump, with peculiar coordination of two water molecules and hydrogen-mediated bonds near the TSD motif, as well as a short B-C loop. Structural and functional analyses of MrHR revealed key residues responsible for the anion selectivity of cyanobacterial halorhodopsin and the involvement of two chloride ion-binding sites in the ion conduction pathway. Alanine mutant of Asn63, Pro118, and Glu182 locating in the anion inlet induce multifunctional uptake of chloride, nitrate, and sulfate ions. Moreover, the structure of N63A/P118A provides information on how SyHR promotes divalent ion transport. Our findings significantly advance the structural understanding of microbial rhodopsins with different motifs. They also provide insight into the general structural framework underlying the molecular mechanisms of the cyanobacterial chloride pump containing SyHR, the only molecule known to transport both sulfate and chloride ions.
Assuntos
Proteínas de Transporte de Ânions/química , Proteínas de Bactérias/química , Cianobactérias/química , Proteínas de Transporte de Ânions/metabolismo , Ânions/metabolismo , Proteínas de Bactérias/metabolismo , Cloretos/metabolismo , Cristalografia por Raios X , Cianobactérias/metabolismo , Halorrodopsinas/química , Halorrodopsinas/metabolismo , Transporte de Íons , Modelos Moleculares , Conformação ProteicaRESUMO
A newly identified microbial rhodopsin, NM-R3, from the marine flavobacterium Nonlabens marinus, was recently shown to drive chloride ion uptake, extending our understanding of the diversity of mechanisms for biological energy conversion. To clarify the mechanism underlying its function, we characterized the crystal structures of NM-R3 in both the dark state and early intermediate photoexcited states produced by laser pulses of different intensities and temperatures. The displacement of chloride ions at five different locations in the model reflected the detailed anion-conduction pathway, and the activity-related key residues-Cys105, Ser60, Gln224, and Phe90-were identified by mutation assays and spectroscopy. Comparisons with other proteins, including a closely related outward sodium ion pump, revealed key motifs and provided structural insights into light-driven ion transport across membranes by the NQ subfamily of rhodopsins. Unexpectedly, the response of the retinal in NM-R3 to photostimulation appears to be substantially different from that seen in bacteriorhodopsin.
Assuntos
Proteínas de Bactérias/química , Canais de Cloreto/química , Luz , Rodopsina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/química , Ativação do Canal Iônico , Modelos Moleculares , Conformação Proteica , Rodopsina/genética , Rodopsina/metabolismo , Relação Estrutura-Atividade , Água/químicaRESUMO
Current anti-hepatitis B virus (HBV) agents, which include nucleos(t)ide analogs and interferons, can significantly suppress HBV infection. However, there are limitations in the therapeutic efficacy of these agents, indicating the need to develop anti-HBV agents with different modes of action. In this study, through a functional cell-based chemical screening, we found that a thiazolidinedione, troglitazone, inhibits HBV infection independently of the compound's ligand activity for peroxisome proliferator-activated receptor γ (PPARγ). Analog analysis suggested chemical moiety required for the anti-HBV activity and identified ciglitazone as an analog having higher anti-HBV potency. Whereas, most of the reported HBV entry inhibitors target viral attachment to the cell surface, troglitazone blocked a process subsequent to viral attachment, i.e., internalization of HBV preS1 and its receptor, sodium taurocholate cotransporting polypeptide (NTCP). We also found that NTCP was markedly oligomerized in the presence of HBV preS1, but such NTCP oligomerization was abrogated by treatment with troglitazone, but not with pioglitazone, correlating with inhibition activity to viral internalization. Also, competitive peptides that blocked NTCP oligomerization impeded viral internalization and infection. This work represents the first report identifying small molecules and peptides that specifically inhibit the internalization of HBV. This study is also significant in proposing a possible role for NTCP oligomerization in viral entry, which will shed a light on a new aspect of the cellular mechanisms regulating HBV infection.
RESUMO
Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial ß-trefoil lectin that recognises Burkitt's lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called "Mitsuba", is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind α-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning "three-leaf" in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Galα(1,4)Galß(1,4)Glc) on the surface, but the cytotoxicity is abolished.
Assuntos
Lectinas/química , Neoplasias/metabolismo , Neoplasias/patologia , Fatores Trefoil/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Morte Celular , Linhagem Celular Tumoral , Biologia Computacional , Cristalografia por Raios X , Hemaglutinação , Humanos , Lectinas/metabolismo , Peso Molecular , Domínios Proteicos , Multimerização Proteica , Coelhos , Açúcares/metabolismoRESUMO
The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.