RESUMO
The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.
Assuntos
Proteínas Fúngicas/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Candida glabrata/química , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Membrana Nuclear/química , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/químicaAssuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Humanos , Antígenos de Histocompatibilidade Menor , Modelos Moleculares , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XAssuntos
Benzoatos/metabolismo , Canais Iônicos/química , Porinas/química , Pseudomonas fluorescens/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Canais Iônicos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Porinas/metabolismo , Conformação Proteica , Homologia de Sequência de AminoácidosAssuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cromatografia em Gel , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
PHR [PAM (protein associated with Myc)-HIW (Highwire)-RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel beta sandwich fold composed of 11 antiparallel beta-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092-->Glu, observed in the Caenorhabditis elegans ortholog RPM-1.
Assuntos
Substituição de Aminoácidos/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Ubiquitina-Proteína LigasesRESUMO
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
