RESUMO
Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 µM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium.
Assuntos
Culicidae , Parasitos , Plasmodium , Animais , Masculino , Actinas/metabolismo , Parasitos/metabolismo , Citoesqueleto de Actina/metabolismo , Culicidae/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium/metabolismoRESUMO
The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza , Fusão de Membrana , Orthomyxoviridae , Internalização do Vírus , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Concentração de Íons de Hidrogênio , Orthomyxoviridae/fisiologia , Conformação ProteicaRESUMO
Malaria is responsible for half a million deaths annually and poses a huge economic burden on the developing world. The mosquito-borne parasites (Plasmodium spp.) that cause the disease depend upon an unconventional actomyosin motor for both gliding motility and host cell invasion. The motor system, often referred to as the glideosome complex, remains to be understood in molecular terms and is an attractive target for new drugs that might block the infection pathway. Here, we present the high-resolution structure of the actomyosin motor complex from Plasmodium falciparum. The complex includes the malaria parasite actin filament (PfAct1) complexed with the class XIV myosin motor (PfMyoA) and its two associated light-chains. The high-resolution core structure reveals the PfAct1:PfMyoA interface in atomic detail, while at lower-resolution, we visualize the PfMyoA light-chain binding region, including the essential light chain (PfELC) and the myosin tail interacting protein (PfMTIP). Finally, we report a bare PfAct1 filament structure at improved resolution.
Assuntos
Malária , Parasitos , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Malária/metabolismo , Miosinas/metabolismo , Parasitos/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the "membrane anchor" portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA-detergent micelles with full-length HA-Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high-resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H1N1/química , Anticorpos Antivirais/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Fragmentos Fab das Imunoglobulinas/química , Micelas , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.
Assuntos
Anticorpos Monoclonais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Hemaglutininas/química , Virus da Influenza A Subtipo H5N1/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Epitopos/química , Humanos , Concentração de Íons de Hidrogênio , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Ligação Proteica , Conformação Proteica , Solventes/químicaRESUMO
Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.
Assuntos
Capsídeo/metabolismo , HIV-1/metabolismo , Modelos Moleculares , Muramidase/química , Proteínas/química , Internalização do Vírus , Sequência de Aminoácidos , Animais , Bacteriófago T4/enzimologia , Sequência de Bases , Cromatografia em Gel , Cristalização , Dimerização , Escherichia coli , Modelos Lineares , Macaca mulatta , Microscopia Eletrônica , Dados de Sequência Molecular , Conformação Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Espalhamento a Baixo Ângulo , Análise de Sequência de DNA , Ressonância de Plasmônio de Superfície , Ubiquitina-Proteína Ligases , Difração de Raios XRESUMO
Images of radiation-sensitive specimens obtained by electron microscopy suffer a reduction in quality beyond that expected from radiation damage alone due to electron beam-induced charging or movement of the specimen. For biological specimens, charging and movement are most severe when they are suspended in an insulating layer of vitreous ice, which is otherwise optimal for preserving hydrated specimens in a near native state. We image biological specimens, including a single particle protein complex and a lipid-enveloped virus in thin, vitreous ice films over suspended sheets of unmodified graphene. We show that in such preparations, the charging of ice, as assessed by electron-optical perturbation of the imaging beam, is eliminated. We also use the same specimen supports to record high resolution images at liquid nitrogen temperature of monolayer paraffin crystals grown over graphene.
Assuntos
Microscopia Crioeletrônica/métodos , Criopreservação/métodos , Grafite/química , Apoferritinas/ultraestrutura , Cristalografia , Microscopia Eletrônica de Transmissão/métodos , Orthomyxoviridae/ultraestrutura , Parafina/química , Vírion/ultraestruturaRESUMO
Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter. We show that the matrix layer adjacent to the membrane is an ordered helix of the M1 protein and its close interaction with the surrounding envelope determines virion morphology. The ribonucleoprotein particles (RNPs) that package the genome segments form a tapered assembly at one end of the virus interior. The neuraminidase, which is present in smaller numbers than the hemagglutinin, clusters in patches and are typically present at the end of the virion opposite to RNP attachment. Incubation of virus at low pH causes a loss of filamentous morphology, during which we observe a structural transition of the matrix layer from its helical, membrane-associated form to a multilayered coil structure inside the virus particle. The polar organization of the virus provides a model for assembly of the virion during budding at the host membrane. Images and tomograms of A/Aichi/68 X-31 virions show the generality of these conclusions to non-filamentous virions.
Assuntos
Vírus da Influenza A/ultraestrutura , Microscopia Crioeletrônica , Concentração de Íons de Hidrogênio , Ribonucleoproteínas/ultraestrutura , Proteínas Virais/ultraestrutura , Vírion/ultraestruturaRESUMO
SARS-CoV-2 is a lipid-enveloped Betacoronavirus and cause of the Covid-19 pandemic. To study the three-dimensional architecture of the virus, we perform electron cryotomography (cryo-ET) on SARS-Cov-2 virions and three variants revealing particles of regular cylindrical morphology. The ribonucleoprotein particles packaging the genome in the virion interior form a dense, double layer assembly with a cylindrical shape related to the overall particle morphology. This organisation suggests structural interactions important to virus assembly.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Pandemias , Elétrons , Microscopia Crioeletrônica/métodos , VírionRESUMO
Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cµ4 and Cµ3 constant regions and the J-chain. A hinge is located at the Cµ3/Cµ2 domain interface, allowing Fabs and Cµ2 to pivot as a unit both in-plane and out-of-plane. This motion is different from that observed in IgG and IgA, where the two Fab arms are able to swing independently. A biased orientation of one pair of Fab arms results from asymmetry in the constant domain (Cµ3) at the IgM subunit interacting most extensively with the J-chain. This may influence the multi-valent binding to surface-associated antigens and complement pathway activation. By comparison, the structure of the Fc fragment in the IgM monomer is similar to that of the pentamer, but is more dynamic in the Cµ4 domain.
Assuntos
Imunoglobulina A , Fragmentos Fc das Imunoglobulinas , Humanos , Imunoglobulina M , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G , Fragmentos Fab das Imunoglobulinas/químicaRESUMO
The lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF's membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.
Assuntos
Gammainfluenzavirus/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Animais , Cães , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Gammainfluenzavirus/ultraestrutura , Células Madin Darby de Rim Canino , Fusão de Membrana , Modelos Moleculares , Multimerização Proteica , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/ultraestruturaRESUMO
Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.
Assuntos
COVID-19/prevenção & controle , Evolução Molecular , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Ligação Competitiva , COVID-19/epidemiologia , COVID-19/virologia , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Pandemias , Pangolins/virologia , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
The oligomeric state and the hydrodynamic properties of human respiratory syncytial virus (HRSV) phosphoprotein (P), a known cofactor of the viral RNA-dependent RNA polymerase (L), and a trypsin-resistant fragment (X) that includes its oligomerization domain were analyzed by sedimentation equilibrium and velocity using analytical ultracentrifugation. The results obtained demonstrate that both P and fragment X are homotetrameric with elongated shapes, consistent with electron micrographs of the purified P protein in which thin rod-like molecules of approximately 12.5 +/- 1.0 nm in length were observed. A new chymotrypsin resistant fragment (Y*) included in fragment X has been identified and purified by gel filtration chromatography. Fragment Y* may represent a minimal version of the P oligomerization domain. Thermal denaturation curves based on circular dichroism data of P protein showed a complex behavior. In contrast, melting data generated for fragments X and particularly fragment Y* showed more homogeneous transitions indicative of simpler structures. A three-dimensional model of X and Y* fragments was built based on the atomic structure of the P oligomerization domain of the related Sendai virus, which is in good agreement with the experimental data. This model will be an useful tool to make rational mutations and test the role of specific amino acids in the oligomerization and functional properties of the HRSV P protein.
Assuntos
Coenzimas/química , RNA Polimerase Dependente de RNA/metabolismo , Vírus Sincicial Respiratório Humano/enzimologia , Homologia de Sequência de Aminoácidos , Proteínas Estruturais Virais/química , Sequência de Aminoácidos , Quimotripsina , Dicroísmo Circular , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Desnaturação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Temperatura , Termodinâmica , Tripsina , Proteínas Estruturais Virais/ultraestruturaRESUMO
BACKGROUND: Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs. RESULTS: Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5-10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach. CONCLUSION: The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies.
Assuntos
Alantoide/metabolismo , Líquidos Corporais/metabolismo , Genoma Viral/genética , Glicoproteínas/biossíntese , Glicoproteínas/genética , Óvulo/metabolismo , Vírus Sendai/genética , Animais , Embrião de Galinha , Galinhas , Cricetinae , Glicoproteínas/isolamento & purificação , Glicoproteínas/ultraestrutura , Humanos , Solubilidade , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/ultraestruturaRESUMO
A novel form of actomyosin regulation has recently been proposed in which the polymerisation of new actin filaments regulates apicomplexan parasite motility. Here, we identified actin I in the merozoites of Plasmodium falciparum by mass spectrometry. The only post-translational modification is acetylation of the N terminus (acetyl-Gly-Glu-actin), while methylation of histidine 73, a common modification for actin, is absent. Results obtained with anti-actin antibodies suggest that, in contrast to a previous report, there is no actin-ubiquitin conjugate in merozoites. About half of the extracted monomeric actin polymerised and actin filaments could be sedimented at 500,000g. In contrast, centrifugation at 100,000g, conditions commonly used to sediment filamentous actin, yielded very little F-actin. In a functional characterisation using an in vitro motility assay, actin filaments moved over myosin at a velocity indistinguishable from that of rabbit skeletal actin. Filament length, however, was too short to be resolved by conventional fluorescence microscopy. On electron micrographs an average filament length of approximately 100nm was determined. We also identified by mass spectrometry proteins co-purifying with filamentous actin, which are potential actin-binding proteins. Our results demonstrate differences in actin filament dynamics for an apicomplexan parasite, which could be due to specific properties of the actin and/or actin-regulatory proteins.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Plasmodium falciparum/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/isolamento & purificação , Animais , Espectrometria de Massas , Microscopia Eletrônica , Plasmodium falciparum/ultraestrutura , Processamento de Proteína Pós-Traducional , Análise de Sequência de ProteínaRESUMO
The Rna14-Rna15 complex is a core component of the cleavage factor IA RNA-processing complex from Saccharomyces cerevisiae. To understand the assembly and RNA-binding properties, we have isolated and characterized the Rna14-Rna15 complex using a combination of biochemical and biophysical methods. Analysis of the purified complex, using transmission electron microscopy, reveals that the two proteins assemble into a kinked rod-shaped structure and that these rods are able to further self-associate. Analytical ultracentrifugation reveals that Rna14 mediates this association and facilitates assembly of an A2B2 tetramer (M(r) 230 000), where relatively compact Rna14-Rna15 heterodimers are in rapid equilibrium with tetramers that have a more extended shape. The Rna14-Rna15 complex, unlike the individual components, binds to an RNA oligonucleotide derived from the 3'-untranslated region of the S.cerevisiae GAL7 gene. Based on these structural and thermodynamic data, we propose that CFIA assembly regulates RNA-binding activity.
Assuntos
RNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Substâncias Macromoleculares , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fatores de Poliadenilação e Clivagem de mRNA/ultraestruturaRESUMO
The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy.
Assuntos
Vírus da Influenza A/ultraestrutura , Internalização do Vírus , Animais , Embrião de Galinha , Microscopia Crioeletrônica , Endossomos/ultraestrutura , Endossomos/virologia , Concentração de Íons de Hidrogênio , Vírus da Influenza A/fisiologia , Lipossomos/química , Fusão de Membrana , Ligação ViralRESUMO
BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.
Assuntos
Proteína BRCA1/química , Proteínas de Transporte/química , Enzimas Desubiquitinantes/química , Histonas/química , Complexos Multiproteicos/química , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/genética , Proteína BRCA1/ultraestrutura , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Cromatina/química , Cromatina/genética , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/ultraestrutura , Instabilidade Genômica , Histonas/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Reparo de DNA por Recombinação/genética , Ubiquitina/genéticaRESUMO
Blood-stage malaria vaccine candidates include surface proteins of the merozoite. Antibodies to these proteins may either block essential steps during invasion or render the merozoite susceptible to phagocytosis or complement-mediated degradation. Structural information on merozoite surface proteins complexed to antibodies provides crucial information for knowledge-based vaccine design. The major merozoite surface protein MSP1 is an abundant surface molecule in Plasmodium falciparum. Only a subset of antibodies against MSP119 inhibits invasion (inhibitory antibodies), whereas other antibodies binding to MSP119 have no effect on invasion (neutral antibodies). Here we report on the complex of MSP119 with both inhibitory monoclonal antibody 12.10 and neutral monoclonal antibody 2F10. The complexes were established using both whole IgG's and Fab fragments, and analysed by dynamic light scattering, electron microscopy and analytical ultra centrifugation. Specific ring structures were formed in the ternary complex with the two antibodies, providing direct evidence of non-overlapping epitopes on MSP119. Mutational studies also indicated that the epitopes of the inhibitory and neutral antibodies are spatially remote.
Assuntos
Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Complexo Antígeno-Anticorpo/química , Proteína 1 de Superfície de Merozoito/química , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Análise Mutacional de DNA , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Proteína 1 de Superfície de Merozoito/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Quaternária de Proteína , UltracentrifugaçãoRESUMO
Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV_F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV_F, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV_F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule.