RESUMO
The ß1-adrenergic receptor (ß1-AR) can activate two families of G proteins. When coupled to Gs, ß1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey ß1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with ß1-AR, the overall interacting modes between ß1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by ß1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Estrutura Terciária de Proteína/fisiologia , Receptores Adrenérgicos beta 1/metabolismo , Animais , Débito Cardíaco/genética , Débito Cardíaco/fisiologia , Linhagem Celular , Microscopia Crioeletrônica , AMP Cíclico/metabolismo , Ativação Enzimática/fisiologia , Células HEK293 , Cardiopatias/patologia , Humanos , Hipertensão/patologia , Isoproterenol/química , Estrutura Secundária de Proteína/fisiologia , Células Sf9 , Transdução de Sinais/fisiologiaRESUMO
Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.
Assuntos
COVID-19/virologia , SARS-CoV-2/fisiologia , Replicação Viral/genética , Monofosfato de Adenosina/farmacologia , Antivirais/farmacologia , COVID-19/genética , COVID-19/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Microscopia Crioeletrônica/métodos , DNA Helicases/metabolismo , Genoma Viral , Humanos , Simulação de Dinâmica Molecular , RNA Helicases/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genéticaRESUMO
SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.
Assuntos
Metiltransferases/química , RNA Helicases/química , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Replicação Viral , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Magnésio/metabolismo , Metiltransferases/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Viral/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/metabolismoRESUMO
The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/classificação , Linfócitos B/citologia , Linfócitos B/metabolismo , Cristalografia por Raios X , Feminino , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/classificação , HIV-1/metabolismo , Humanos , Macaca mulatta , Masculino , Peptídeos/química , Estrutura Terciária de Proteína , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Animais , Feminino , Cobaias , HIV-1/efeitos dos fármacos , Imunização , Macaca mulatta , Camundongos Endogâmicos C57BL , Modelos Moleculares , Testes de Neutralização , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin α(IIb)ß(3) to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-ß and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.
Assuntos
Fator de von Willebrand/química , Fator de von Willebrand/genética , Sequência de Aminoácidos , Sítios de Ligação , Dimerização , Humanos , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Fator de von Willebrand/ultraestruturaRESUMO
At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal â¼1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.