Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
1.
Nat Struct Mol Biol ; 16(3): 238-46, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19198587

RESUMO

The AU-rich element (ARE)-mediated mRNA-degradation activity of the RNA binding K-homology splicing regulator protein (KSRP) is regulated by phosphorylation of a serine within its N-terminal KH domain (KH1). In the cell, phosphorylation promotes the interaction of KSRP and 14-3-3zeta protein and impairs the ability of KSRP to promote the degradation of its RNA targets. Here we examine the molecular details of this mechanism. We report that phosphorylation leads to the unfolding of the structurally atypical and unstable KH1, creating a site for 14-3-3zeta binding. Using this site, 14-3-3zeta discriminates between phosphorylated and unphosphorylated KH1, driving the nuclear localization of KSRP. 14-3-3zeta -KH1 interaction regulates the mRNA-decay activity of KSRP by sequestering the protein in a separate functional pool. This study demonstrates how an mRNA-degradation pathway is connected to extracellular signaling networks through the reversible unfolding of a protein domain.


Assuntos
Proteínas 14-3-3/metabolismo , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Alinhamento de Sequência
5.
Structure ; 14(1): 21-31, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16407062

RESUMO

FMRP, whose lack of expression causes the X-linked fragile X syndrome, is a modular RNA binding protein thought to be involved in posttranslational regulation. We have solved the structure in solution of the N-terminal domain of FMRP (NDF), a functionally important region involved in multiple interactions. The structure consists of a composite fold comprising two repeats of a Tudor motif followed by a short alpha helix. The interactions between the three structural elements are essential for the stability of the NDF fold. Although structurally similar, the two repeats have different dynamic and functional properties. The second, more flexible repeat is responsible for interacting both with methylated lysine and with 82-FIP, one of the FMRP nuclear partners. NDF contains a 3D nucleolar localization signal, since destabilization of its fold leads to altered nucleolar localization of FMRP. We suggest that the NDF composite fold determines an allosteric mechanism that regulates the FMRP functions.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/química , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Regulação Alostérica/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/fisiologia , Humanos , Lisina/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Mutação Puntual , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA
6.
Genes Dev ; 19(12): 1444-54, 2005 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15933069

RESUMO

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Drosophila/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/metabolismo , Humanos , Técnicas In Vitro , Metilação , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
7.
EMBO J ; 24(14): 2491-8, 2005 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-15973432

RESUMO

The secreted Mycobacterium tuberculosis complex proteins CFP-10 and ESAT-6 have recently been shown to play an essential role in tuberculosis pathogenesis. We have determined the solution structure of the tight, 1:1 complex formed by CFP-10 and ESAT-6, and employed fluorescence microscopy to demonstrate specific binding of the complex to the surface of macrophage and monocyte cells. A striking feature of the complex is the long flexible arm formed by the C-terminus of CFP-10, which was found to be essential for binding to the surface of cells. The surface features of the CFP-10.ESAT-6 complex, together with observed binding to specific host cells, strongly suggest a key signalling role for the complex, in which binding to cell surface receptors leads to modulation of host cell behaviour to the advantage of the pathogen.


Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Transdução de Sinais/fisiologia , Fatores de Virulência/química , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Células COS , Linhagem da Célula , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Humanos , Camundongos , Dados de Sequência Molecular , Monócitos/metabolismo , Células NIH 3T3 , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Células U937 , Fatores de Virulência/fisiologia
9.
Biochemistry ; 44(2): 518-23, 2005 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-15641776

RESUMO

We have applied NMR cross-saturation with TROSY detection to the problem of precisely mapping conformational epitopes on complete protein antigen molecules. We have investigated complexes of the Fab fragments of two antibodies that have parasite inhibitory activity, bound to the important malaria vaccine candidate antigen, Plasmodium falciparum MSP1(19). The results indicate remarkable overlap between these epitopes for inhibitory antibodies, and will provide a basis for theoretical modeling of the antibody-antigen interface.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Antiprotozoários/química , Antimaláricos/química , Sítios de Ligação de Anticorpos , Mapeamento de Epitopos , Epitopos Imunodominantes/química , Proteína 1 de Superfície de Merozoito/química , Plasmodium falciparum/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Antimaláricos/metabolismo , Mapeamento de Epitopos/métodos , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteína 1 de Superfície de Merozoito/imunologia , Proteína 1 de Superfície de Merozoito/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Plasmodium falciparum/crescimento & desenvolvimento , Conformação Proteica , Sensibilidade e Especificidade , Soluções , Ressonância de Plasmônio de Superfície/métodos
11.
Mol Biochem Parasitol ; 138(1): 29-36, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15500913

RESUMO

Plasmodium falciparum merozoite surface protein 1 (MSP1)(19), the C-terminal fragment of merozoite surface protein 1, is a leading candidate antigen for development of a vaccine against the blood stages of the malaria parasite. Many human and animal studies have indicated the importance of MSP1(19)-specific immune responses. Anti-MSP1(19) antibodies can prevent invasion of red blood cells by P. falciparum parasites in vitro. However, the fine specificity of anti-MSP1(19) antibodies is also important, as only a fraction of monoclonal antibodies (mAbs) have parasite-inhibitory activity in vitro. Human sera from malaria-endemic locations show strong MSP1(19) reactivity, but individual serum samples vary greatly in inhibitory activity. NMR is an excellent method for studying protein-protein interactions, and has been used widely to study binding of peptides representing known epitopes (as well as non-protein antigens) to antibodies and antibody fragments. The recent development of transverse relaxation optimized spectroscopy (TROSY) and related methods has significantly extended the maximum size limit of molecules that can be studied by NMR. TROSY NMR experiments produce high quality spectra of Fab complexes that allow the mapping of epitopes by the chemical shift perturbation technique on a complete, folded protein antigen such as MSP1(19). We studied the complexes of P. falciparum MSP1(19) with Fab fragments from three monoclonal antibodies. Two of these antibodies have parasite-inhibitory activity in vitro, while the third is non-inhibitory. NMR epitope mapping showed a close relationship between binding sites for the two inhibitory antibodies, distinct from the location of the non-inhibitory antibody. Together with a previously published crystal structure of the P. falciparum MSP1(19) complex with the Fab fragment of another non-inhibitory antibody, these results revealed a surface on MSP1(19) where inhibitory antibodies bind. This information will be useful in evaluating the anti-MSP1(19) immune response in natural populations from endemic areas, as well as in vaccine trials. It will also be valuable for optimizing the MSP1(19) antigen by rational vaccine design. This work also shows that TROSY NMR techniques are very effective for mapping conformational epitopes at the level of individual residues on small- to medium-sized proteins, provided that the antigen can be expressed in a system amenable to stable isotope labelling, such as bacteria or yeast.


Assuntos
Anticorpos Antiprotozoários/imunologia , Mapeamento de Epitopos/métodos , Espectroscopia de Ressonância Magnética/métodos , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Animais , Anticorpos Antiprotozoários/metabolismo , Reações Antígeno-Anticorpo , Epitopos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Malária Falciparum/imunologia , Proteína 1 de Superfície de Merozoito/metabolismo , Modelos Moleculares , Dados de Sequência Molecular
15.
FEBS Lett ; 549(1-3): 21-5, 2003 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-12914917

RESUMO

Anomalous expansion of a polyglutamine (polyQ) tract in the protein ataxin-3 causes spinocerebellar ataxia type 3, an autosomal dominant neurodegenerative disease. Very little is known about the structure and the function of ataxin-3, although this information would undoubtedly help to understand why the expanded protein forms insoluble nuclear aggregates and causes neuronal cell death. With the aim of establishing the domain architecture of ataxin-3 and the role of the polyQ tract within the protein context, we have studied the human and murine orthologues using a combination of techniques, which range from limited proteolysis to circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The two protein sequences share a highly conserved N-terminus and differ only in the length of the glutamine repeats and in the C-terminus. Our data conclusively indicate that ataxin-3 is composed by a structured N-terminal domain, followed by a flexible tail. Moreover, [(15)N]glutamine selectively labelled samples allowed us to have a direct insight by NMR into the structure of the polyQ region.


Assuntos
Proteínas do Tecido Nervoso/química , Sequência de Aminoácidos , Animais , Ataxina-3 , Dicroísmo Circular , Humanos , Camundongos , Dados de Sequência Molecular , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares , Peptídeo Hidrolases/metabolismo , Peptídeos , Estrutura Terciária de Proteína , Proteínas Repressoras , Alinhamento de Sequência , Fatores de Transcrição
16.
J Biol Chem ; 278(44): 43736-43, 2003 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-12917404

RESUMO

The closely related mycobacteria responsible for tuberculosis produce an unusually high number of secreted proteins, many of which are clearly implicated in pathogenesis and protective immunity. Falling within this category are the closely related proteins MPB70 and MPB83. The structure of MPB70 reveals a complex and novel bacterial fold, which has clear structural homology to the two C-terminal FAS1 domains of the cell adhesion protein fasciclin I, whose structures were reported very recently. Assessment of the surface features of MPB70, the sequence divergence between MPB70 and MPB83, the conservation of residues across a group of FAS1 domains, and the locations of disease-inducing mutations in betaig-h3 strongly suggests that MPB70 and MPB83 contain two functional surfaces on opposite faces, which are probably involved in binding to host cell proteins. This analysis also suggests that these functional surfaces are retained in the FAS1 proteins associated with mediating interactions between cells and the extracellular matrix (fasciclin I, periostin, and betaig-h3) and furthermore that some of the human corneal disease-inducing substitutions identified in betaig-h3 will perturb interactions at these sites.


Assuntos
Proteínas de Bactérias/química , Doenças da Córnea/metabolismo , Mycobacterium tuberculosis/metabolismo , Fator de Crescimento Transformador beta , Tuberculose/microbiologia , Sequência de Aminoácidos , Antígenos de Bactérias/química , Proteínas de Bactérias/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular Neuronais/química , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
17.
Biochemistry ; 42(5): 1266-73, 2003 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-12564929

RESUMO

The three-dimensional solution structure of the DNA-binding domain of Mlu-1 box binding protein (Mbp1) has been determined by multidimensional NMR spectroscopy. Mbp1 is a cell cycle transcription factor from Saccharomyces cerevisiae and consists of an N-terminal DNA-binding domain, a series of ankyrin repeats, and a heterodimerization domain at the C-terminus. A set of conformers comprising 19 refined structures was calculated via a molecular dynamics simulated annealing protocol using distance, dihedral angle, and residual dipolar coupling restraints derived from either double or triple resonance NMR experiments. The solution structure consists of a six-stranded beta-sheet segment folded against two pairs of alpha-helices in the topology of the winged helix-turn-helix family of proteins and is in agreement with the X-ray structures. In addition, the solution structure shows that the C-terminal tail region of this domain folds back and makes specific interactions with the N-terminal beta-strand of the protein. This C-terminal region is essential for full DNA-binding activity but appears in the X-ray structure to be disordered. The fold-back structure extends the region of positive electrostatic potential, and this may enhance the nonspecific contribution to binding by favorable electrostatic interactions with the DNA backbone.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Ressonância Magnética Nuclear Biomolecular , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Simulação por Computador , Cristalografia por Raios X , Sequências Hélice-Volta-Hélice , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções , Eletricidade Estática , Termodinâmica
18.
Chembiochem ; 3(11): 1072-7, 2002 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-12404632

RESUMO

NKR-P1A is a C-type lectin-like receptor on natural killer cells believed to be involved in the cytotoxicity of these cells. Ligands for this protein are not known. Here, we describe the binding of a fully sulphated disaccharide, sucrose octasulphate, by the recombinant C-type lectin-like domain of NKR-P1A. The binding was observed by NMR spectroscopy methods that have recently been described for the screening of compound libraries for bioaffinities, namely the 2D NOESY and saturation transfer difference NMR experiments. (1)H titration studies indicate that the binding is specific. These findings raise the possibility that NKR-P1A recognises sulphated natural ligands in common with certain other members of the C-type lectin family.


Assuntos
Antígenos de Superfície/química , Lectinas Tipo C/química , Sacarose/análogos & derivados , Sacarose/química , Humanos , Células Matadoras Naturais/química , Ligantes , Espectroscopia de Ressonância Magnética , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...