Assuntos
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
Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
Assuntos
Regulação da Expressão Gênica em Archaea/genética , Ácidos Cetoglutáricos/metabolismo , Mathanococcus/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas PII Reguladoras de Nitrogênio/química , Conformação Proteica , Fatores de Transcrição/química , Microscopia Eletrônica , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.