RESUMO
Arabidopsis thaliana Deg8, an ATP-independent serine endopeptidase, is involved in the repair of photosystem II (PSII), specifically the degradation of the photo-damaged PSII reaction centre D1 protein. To understand the molecular mechanism underlying the participation of Deg8 in the degradation of the photo-damaged D1 protein, the structure of Deg8 is needed. Until recently, however, no structure of Deg8 had been solved. In this study, Deg8 from A. thaliana was cloned, overexpressed and purified in Escherichia coli. Crystallization was performed at 277â K using tribasic sodium citrate as the precipitant and the crystals diffracted to 2.0â Å resolution, belonging to space group C2 with unit-cell parameters a = 129.5, b = 124.2, c = 93.3â Å, α = γ = 90, ß = 132.4°. Assuming one trimer in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.35â Å(3)â Da(-1) and 47.6%, respectively.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Serina Endopeptidases/química , Serina Endopeptidases/isolamento & purificação , Proteínas de Arabidopsis/genética , Sequência de Bases , Clonagem Molecular , Cristalização/métodos , Cristalografia por Raios X , Dados de Sequência Molecular , Mutação , Conformação Proteica , Serina Endopeptidases/genéticaRESUMO
The flavoenzyme fructosyl peptide oxidase (FPOX) catalyses the oxidative deglycation of fructosyl amino acids or fructosyl dipeptides to produce amino acids, glucosone and hydrogen peroxide. In this study, FPOX protein from Eupenicillium terrenum sp. (EtFPOX) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. EtFPOX crystals were obtained using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 65.6, b = 80.0, c = 83.4 Å, and contained one molecule in the asymmetric unit. The calculated Matthews coefficient and solvent content were 2.22 Å(3) Da(-1) and 44.62%, respectively.
Assuntos
Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/química , Eupenicillium/enzimologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Aminoácido Oxirredutases/isolamento & purificação , Cristalização , Proteínas Fúngicas/isolamento & purificação , Difração de Raios XRESUMO
Thioesterase superfamily member 2 (THEM2) is essential for cell proliferation of mammalian cells. It belongs to the hotdog-fold thioesterase superfamily and catalyzes the hydrolysis of the thioester bonds of acyl-CoA in vitro. In this study, THEM2 protein from zebrafish (fTHEM2) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. fTHEM2 crystals were obtained using the sitting-drop vapour-diffusion method with PEG 10â 000 as precipitant. X-ray diffraction data were collected to 1.80â Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=77.1, b=74.4, c=96.6â Å, ß=93.7°.
Assuntos
Palmitoil-CoA Hidrolase/química , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/isolamento & purificação , Peixe-Zebra/metabolismo , Acil Coenzima A/química , Animais , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Palmitoil-CoA Hidrolase/genética , Palmitoil-CoA Hidrolase/isolamento & purificação , Difração de Raios X , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
We report a 3.5-angstrom-resolution cryo-electron microscopy structure of a respiratory supercomplex isolated from Mycobacterium smegmatis. It comprises a complex III dimer flanked on either side by individual complex IV subunits. Complex III and IV associate so that electrons can be transferred from quinol in complex III to the oxygen reduction center in complex IV by way of a bridging cytochrome subunit. We observed a superoxide dismutase-like subunit at the periplasmic face, which may be responsible for detoxification of superoxide formed by complex III. The structure reveals features of an established drug target and provides a foundation for the development of treatments for human tuberculosis.
Assuntos
Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Transporte de Elétrons , Mycobacterium smegmatis/enzimologia , Superóxido Dismutase/química , Actinobacteria/enzimologia , Microscopia Crioeletrônica , Oxirredução , Fosforilação Oxidativa , Oxigênio/metabolismo , Multimerização ProteicaAssuntos
Fator de Iniciação Eucariótico 4G/química , Proteínas do Tecido Nervoso/química , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Cristalografia por Raios X , Bases de Dados de Proteínas , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Biblioteca Gênica , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Selenometionina/química , Selenometionina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Propriedades de SuperfícieRESUMO
The FAOD/FPOD family of proteins has the potential to be useful for the longterm detection of blood glucose levels in diabetes patients. A bottleneck for this application is to find or engineer a FAOD/FPOD family enzyme that is specifically active towards α-fructosyl peptides but is inactive towards other types of glycated peptides. Here, the crystal structure of fructosyl peptide oxidase from Eupenicillium terrenum (EtFPOX) is reported at 1.9â Å resolution. In contrast to the previously reported structure of amadoriase II, EtFPOX has an open substrate entrance to accommodate the large peptide substrate. The functions of residues critical for substrate selection are discussed based on structure comparison and sequence alignment. This study reveals the first structural details of group I FPODs that prefer α-fructosyl substrates and could provide significant useful information for uncovering the mechanism of substrate specificity of FAOD/FPODs and guidance towards future enzyme engineering for diagnostic purposes.
Assuntos
Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Eupenicillium/enzimologia , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato/fisiologiaRESUMO
The C-terminal nuclear localization sequence of FUsed in Sarcoma (FUS-NLS) is critical for its nuclear import mediated by transportin (Trn1). Familial amyotrophic lateral sclerosis (ALS) related mutations are clustered in FUS-NLS. We report here the structural, biochemical and cell biological characterization of the FUS-NLS and its clinical implications. The crystal structure of the FUS-NLS/Trn1 complex shows extensive contacts between the two proteins and a unique α-helical structure in the FUS-NLS. The binding affinity between Trn1 and FUS-NLS (wide-type and 12 ALS-associated mutants) was determined. As compared to the wide-type FUS-NLS (K(D) = 1.7 nM), each ALS-associated mutation caused a decreased affinity and the range of this reduction varied widely from 1.4-fold over 700-fold. The affinity of the mutants correlated with the extent of impaired nuclear localization, and more importantly, with the duration of disease progression in ALS patients. This study provides a comprehensive understanding of the nuclear targeting mechanism of FUS and illustrates the significance of FUS-NLS in ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/metabolismo , beta Carioferinas/química , beta Carioferinas/metabolismo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteína FUS de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , beta Carioferinas/genéticaRESUMO
Eukaryotic translation initiation factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2, catalyzes conversion of eIF2·GDP to eIF2·GTP. The eIF2B is composed of five subunits, α, ß, γ, δ and É, within which the É subunit is responsible for catalyzing the guanine exchange reaction. Here we present the crystal structure of the C-terminal domain of human eIF2BÉ (eIF2BÉ-CTD) at 2.0-Å resolution. The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified. When compared to yeast eIF2BÉ-CTD, one area involves highly conserved AA boxes while the other two are only partially conserved. In addition, the previously reported mutations in human eIF2BÉ-CTD, which are related to the loss of the GEF activity and human VWM disease, have been discussed. Based on the structure, most of such mutations tend to destabilize the HEAT motif.