RESUMO
The encapsulation of otherwise transcribable loci within transcriptionally inactive heterochromatin is rapidly gaining recognition as an important mechanism of epigenetic gene regulation. In the fission yeast Schizosaccharomyces pombe, heterochromatinization of the mat2/mat3 loci silences the mating-type information encoded within these loci. Here, we present the solution structure of the chromo domain from the cryptic loci regulator protein Clr4. Clr4 is known to regulate silencing and switching at the mating-type loci and to affect chromatin structure at centromeres. Clr4 and its human and Drosophila homologs have been identified as histone H3-specific methyltransferases, further implicating this family of proteins in chromatin remodeling. Our structure highlights a conserved surface that may be involved in chromo domain-ligand interactions. We have also analyzed two chromo domain mutants (W31G and W41G) that previously were shown to affect silencing and switching in full-length Clr4. Both mutants are significantly destabilized relative to wild-type.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase , Metiltransferases/química , Metiltransferases/metabolismo , Mutação/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Sequência Conservada , Inativação Gênica , Histona Metiltransferases , Metiltransferases/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas Metiltransferases , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Schizosaccharomyces/genética , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
The complex and interrelated function of the interleukin cytokines relies on a range of pro-inflammatory and anti-inflammatory immune responses mediated by an array of receptors, and there is considerable cross-reactivity for related cytokines. Recent findings continue to elucidate the expression patterns of interleukin receptors associated with a range of diseases, including cancer. We report here the first experimentally determined high-resolution structure of human interleukin-13 (IL-13). The experimental structure is significantly different from an earlier homology model, which could have led to improper estimation of receptor interaction surfaces and design of mutational experiments. Similarities between the presented IL-13 structure and the homologous interleukin-4 (IL-4) are discussed. Additionally, mutation data for IL-4 and IL-13 are analyzed and combined with a detailed structural analysis of the IL-4/IL4Ralpha interface that leads us to postulate interactions at the IL-13/receptor interface. The structural comparison is used to interpret the different affinities for various receptors and establishes the basis for further mutational experiments and antagonist design.
Assuntos
Interleucina-13/química , Interleucina-13/metabolismo , Ressonância Magnética Nuclear Biomolecular , Receptores de Interleucina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Subunidade alfa1 de Receptor de Interleucina-13 , Interleucina-2/química , Interleucina-2/metabolismo , Interleucina-4/química , Interleucina-4/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores de Interleucina-13 , Receptores de Interleucina-4/química , Receptores de Interleucina-4/metabolismo , Alinhamento de Sequência , Soluções , Eletricidade EstáticaRESUMO
SH3 domains are protein binding domains that occur widely among signal transduction proteins. Here, we present the NMR-determined solution structure of the SH3 domain from the cytoplasmic protein tyrosine kinase, Hck. Hck is involved in a number of cell signal transduction pathways, frequently in pathways associated with immune response. SH3 domains bind proteins via a left-handed polyproline type II helix on the target protein. We have assessed the structural impact of binding to a ligand through addition of a peptide corresponding to a proline-rich region of a Hck target, the GTPase activating protein of the Ras pathway. Ligand binding effects small structural changes and stabilizes the SH3 domain structure. Also, we have compared the solution structure of the Hck SH3 domain to the crystal structure of Hck, in which the SH3 domain exhibits an intramolecular binding to an interdomain linker region. These structures are interpreted as the apo- and holo- forms of the Hck SH3 domain.
Assuntos
Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/química , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Proto-Oncogênicas c-hck , SoluçõesRESUMO
The NusB protein is involved in transcriptional regulation in bacteriophage lambda. NusB binds to the RNA form of the nut site and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg-rich RNA-binding motif (ARM) at the N-terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM-containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six alpha-helices: R10-Q21, 127-F34, V45-L65, Q79-S93, Y100-F114 and D118-L127. The structure of NusB makes it a member of a newly emerging class of alpha-helical RNA-binding proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Dobramento de Proteína , Proteínas Recombinantes/químicaAssuntos
Bacteriófago lambda/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Nucleocapsídeo/metabolismo , RNA Viral/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Gênica , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/genética , Sequência de Bases , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Indicadores e Reagentes , Cinética , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleocapsídeo/isolamento & purificação , Óperon , Fatores de Alongamento de Peptídeos/isolamento & purificação , Fatores de Alongamento de Peptídeos/metabolismo , Radioisótopos de Fósforo , Regiões Promotoras Genéticas , RNA Viral/análise , RNA Viral/química , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Regiões Terminadoras Genéticas , Fatores de Transcrição/isolamento & purificação , Fatores de Elongação da TranscriçãoRESUMO
We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli. The structure reveals a novel, all alpha-helical protein fold. NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions. Residues that are highly conserved among homologs stabilize the protein core. The solution structure of E. coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E. coli NusB reported earlier.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Escherichia coli/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Mycobacterium tuberculosis/química , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Soluções , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A nonanucleotide, d(G1G2T3C4[BaP]A5C6G7A8G9), in which (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (7-hydroxyl group and epoxide oxygen are trans) is covalently bonded to the exocyclic N6-amino group of deoxyadenosine (dA5) through trans addition at C10 of the epoxide (to give a 10S adduct) has been synthesized. The solution structure of the duplex, d(G1G2T3C4[BaP]A5C6G7A8G9).d(C10T11C12G13G14G15A16C17C18+ ++), containing a dG mismatch opposite the modified dA (designated 10S-[BaP]dA.dG 9-mer duplex) has been investigated using a combination of 1D and 2D (including COSY, PECOSY, TOCSY, NOESY, and indirect detection of 1H-31P HETCOR) NMR spectroscopies. The NMR results together with restrained molecular dynamics/energy minimization calculations show that the modified dA5 adopts a syn glycosidic torsion angle whereas all other nucleotide residues adopt anti glycosidic torsion angles. The sugar ring of dA5 is in the C3'-endo conformation, and the sugar rings of the other residues are in the C2'-endo conformation. The hydrocarbon attached at dA5 orients toward the 3' end of the modified strand (i.e., dC6 direction) and intercalates between and parallel to bases of dG13 and dG14 of the complementary strand directly opposite dC6 and dA5, respectively. The edge of the hydrocarbon bearing H11 and H12 is positioned between the imino protons of dG13 and dG14 in the interior of the duplex, whereas H4 and H5 at the opposite edge are positioned near the sugar H1' and H2" protons of dG13 and facing the exterior of the duplex. The mismatched AG base pair is stabilized by dAsyn-dGanti base pairing in which the imino proton and the O6 of dG14 are hydrogen bonded to N7- and the single N6-amino proton, respectively, of the modified dA5. The modified DNA duplex remains in a right-handed helix, which bends at the site of intercalation about 20 to 30 degrees away from the helical axis and toward the direction of the modified strand.