RESUMO
BACKGROUND: Nucleoside diphosphate (NDP) kinases provide precursors for DNA and RNA synthesis. In mammals, these enzymes are also involved in cell regulations. Human NDP kinase B, product of the human nm23-H2 gene, is both an enzyme and a transcription factor. It activates transcription of the c-myc oncogene independently of its catalytic function, by binding to its promoter DNA. How do the two functions coexist? RESULTS: Recombinant human NDP kinase B was co-crystallized with GDP. The X-ray structure was solved at 2.0 A resolution by molecular replacement from the homologous Drosophila Awd protein. Both enzymes are homo-hexamers with a characteristic beta alpha beta beta alpha beta fold. GDP binds near the active site His118. The guanine base is in a surface cleft and interacts with the C terminus of another subunit. CONCLUSIONS: The beta alpha beta beta alpha beta fold, also present in the 'palm' domain of Escherichia coli DNA polymerase I and HIV reverse transcriptase, is both a mononucleotide- and a polynucleotide-binding fold. If NDP kinase B binds DNA in the same way as the polymerases, the enzyme must undergo a conformation change in order to carry out gene activation.
Assuntos
Proteínas de Drosophila , Guanosina Difosfato/química , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase/química , Conformação Proteica , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , DNA Polimerase I/química , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica , Guanosina Difosfato/metabolismo , Humanos , Hormônios de Inseto/química , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/metabolismo , Ligação Proteica , Multimerização Proteica , DNA Polimerase Dirigida por RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The X-ray structure of nucleoside diphosphate kinase (NDP kinase) from the slime mold Dictyostelium discoideum has been determined to 2.2-A resolution and refined to an R-factor of 0.19 with and without bound ADP-Mg2+. The nucleotide binds near His 122, a residue which becomes phosphorylated during the catalytic cycle. The mode of binding is different from that observed in other phosphokinases, and it involves no glycine-rich sequence. The adenine base makes only nonpolar contacts with the protein. It points outside, explaining the lack of specificity of NDP kinase toward the base. The ribose 2'- and 3'-hydroxyls and the pyrophosphate moiety are H-bonded to polar side chains. A Mg2+ ion bridges the alpha- to the beta-phosphate which approaches the imidazole group of His 122 from the N delta side. The geometry at the active site in the ADP-Mg2+ complex suggests a mechanism for catalysis whereby the gamma-phosphate of a nucleoside triphosphate can be transferred onto His 122 with a minimum of atomic motion.
Assuntos
Difosfato de Adenosina/metabolismo , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Difosfato Quinase/metabolismo , Animais , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dictyostelium/enzimologia , Escherichia coli/enzimologia , Histidina/metabolismo , Substâncias Macromoleculares , Magnésio/metabolismo , Modelos Moleculares , Estrutura Molecular , Fosforilação , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Nucleoside diphosphate kinase from the slime mold Dictyostelium discoideum is highly homologous to gene products that are involved in development in Drosophila and in oncogenesis in human cells. The cloned protein expressed in Escherichia coli has been purified and crystallized in a hexagonal space group with a = b = 74.9 A, c = 211.4 A. The asymmetric unit contains either one or two 17,000 Mr subunits of the hexamer.