Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA.

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (approximately 1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.     

Wild-type NM23-H1, but not its S120 mutants, suppresses desensitization of muscarinic potassium current

NM23 (NDP kinase) modulates the gating of muscarinic K+ channels by agonists through a mechanism distinct from GTP regeneration. To better define the function of NM23 in this pathway and to identify sites in NM23 that are important for its role in muscarinic K+ channel function, we utilized MDA-MB-435 human breast carcinoma cells that express low levels of NM23-H1. M2 muscarinic receptors and GIRK1/GIRK4 channel subunits were co-expressed in cells stably transfected with vector only (control), wild-type NM23-H1, or several NM23-H1 mutants. Lysates from all cell lines tested exhibit comparable nucleoside diphosphate (NDP) kinase activity. Whole cell patch clamp recordings revealed a substantial reduction of the acute desensitization of muscarinic K+ currents in cells overexpressing NM23-H1. The mutants NM23-H1P96S and NM23-H1S44A resembled wild-type NM23-H1 in their ability to reduce desensitization. In contrast, mutants NM23-H1S120G and NM23-H1S120A completely abolished the effect of NM23-H1 on desensitization of muscarinic K+ currents. Furthermore, NM23-H1S120G potentiated acute desensitization, indicating that this mutant retains the ability to interact with the muscarinic pathway, but has properties antithetical to those of the wild-type protein. We conclude that NM23 acts as a suppressor of the processes leading to the desensitization of muscarinic K+ currents, and that Ser-120 is essential for its actions.     

Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis,and the nucleosome assembly protein SET is its inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.     

Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.     

Mechanism of phosphoryl transfer by nucleoside diphosphate kinase pH dependence and role of the active site Lys16 and Tyr56 residues.

Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide analogues used in antiviral therapies are also metabolized to their triphosphate form by NDP kinase, their lack of the 3'-hydroxyl of the ribose, which allows them to be DNA chain terminators, severely impairs the catalytic efficiency of NDP kinase. We have analyzed the kinetics parameters of several mutant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting with the gamma-phosphate and/or the 3'-OH of the Mg2+-ATP substrate. We compared the relative contributions of the active-site residues and the substrate 3'-OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of additional data from pH profiles identify the ionization state of these residues in the enzyme active form. X-ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.     

NMR studies on the flexibility of nucleoside diphosphate kinase

Human NDP kinase B, product of the nm23-H2 gene, binds DNA. It has been suggested that a helix hairpin on the protein surface, part of the nucleotide substrate binding site, could accommodate DNA binding by swinging away. The presence of flexible regions was therefore investigated by ¹H NMR dynamic filtering. Although TOCSY peaks could be assigned to five residues at the N terminus of Dictyostelium NDP kinase, no flexible region was detected in the human enzyme. These data favor the idea that the protein offers different binding sites to mono- and polynucleotides. Proteins 28:150–152, 1997. © 1997 Wiley-Liss Inc.     

Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes

The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.     

NM23-NDP kinase

NM23 belongs to a large family of structurally and functionally conserved proteins consisting of 4–6 identically folded subunits of approximately 16–20 kDa. These oligomeric proteins exhibit nucleoside diphosphate kinase (NDPK) activity that catalyzes nonsubstrate specific conversions of nucleoside diphosphates to nucleoside triphosphates. Many NM23 proteins bind DNA. In vivo, NM23–NDPKs regulate a diverse array of cellular events including growth and development. They are also implicated in the pathogenesis and metastasis of tumors. The mechanism whereby NM23 regulates gene expression is proposed to entail DNA-binding and subsequent alterations in promoter DNA structure. Accordingly, NM23 has the potential to become a useful reagent for gene manipulations.     

The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.     

Experimental and computational investigations of Ser10 and Lys13 in the binding and cleavage of DNA substrates by Escherichia coli DNA topoisomerase I

Ser10 and Lys13 found near the active site tyrosine of Escherichia coli DNA topoisomerase I are conserved among the type IA topoisomerases. Site-directed mutagenesis of these two residues to Ala reduced the relaxation and DNA cleavage activity, with a more severe effect from the Lys13 mutation. Changing Ser10 to Thr or Lys13 to Arg also resulted in loss of DNA cleavage and relaxation activity of the enzyme. In simulations of the open form of the topoisomerase–DNA complex, Lys13 interacts directly with Glu9 (proposed to be important in the catalytic mechanism). This interaction is removed in the K13A mutant, suggesting the importance of lysine as either a proton donor or a stabilizing cation during strand cleavage, while the Lys to Arg mutation significantly distorts catalytic residues. Ser10 forms a direct hydrogen bond with a phosphate group near the active site and is involved in direct binding of the DNA substrate; this interaction is disturbed in the S10A and S10T mutants. This combination of a lysine and a serine residue conserved in the active site of type IA topoisomerases may be required for correct positioning of the scissile phosphate and coordination of catalytic residues relative to each other so that DNA cleavage and subsequent strand passage can take place.     

Crystal structure of fosfomycin resistance kinase FomA from Streptomyces wedmorensis

The fosfomycin resistance protein FomA inactivates fosfomycin by phosphorylation of the phosphonate group of the antibiotic in the presence of ATP and Mg(II). We report the crystal structure of FomA from the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis in complex with diphosphate and in ternary complex with the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP), Mg(II), and fosfomycin, at 1.53 and 2.2 angstroms resolution, respectively. The polypeptide exhibits an open alphabetaalpha sandwich fold characteristic for the amino acid kinase family of enzymes. The diphosphate complex shows significant disorder in loops surrounding the active site. As a result, the nucleotide-binding site is wide open. Binding of the substrates is followed by the partial closure of the active site and ordering of the alpha2-helix. Structural comparison with N-acetyl-L-glutamate kinase shows several similarities in the site of phosphoryl transfer: 1) preservation of architecture of the catalytical amino acids of N-acetyl-L-glutamate kinase (Lys9, Lys216, and Asp150 in FomA); 2) good superposition of the phosphate acceptor groups of the substrates, and 3) good superposition of the diphosphate molecule with the beta- and gamma-phosphates of AMPPNP, suggesting that the reaction could proceed by an associative in-line mechanism. However, differences in conformations of the triphosphate moiety of AMPPNP molecules, the long distance (5.1 angstroms) between the phosphate acceptor and donor groups in FomA, and involvement of Lys18 instead of Lys9 in binding with the gamma-phosphate may indicate a different reaction mechanism. The present work identifies the active site residues of FomA responsible for substrate binding and specificity and proposes their roles in catalysis.     

Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.