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.     

Solution studies on heme proteins: subunit structure, dissociation, and unfolding of Lumbricus terrestris hemoglobin by the ureas.

The subunit structure, dissociation, and unfolding of the hemoglobin of the earthworm, Lumbricus terrestris, were investigated by light scattering molecular weight methods and changes in optical rotatory dispersion (at 233 nm) and absorption in the Soret region. Urea and the alkylureas, methyl-, ethyl-, propyl-, and butylurea, were employed as the reagents to cause both dissociation and unfolding of the protein. Analysis of the light scattering data suggests that the dissociation patterns as a function of hemoglobin concentration in the various dissociating solvents can be described in quantitative terms, either as an equilibrium mixture consisting of parent duodecamers and hexamers of 3 x 10(6) and 1.5 x 10(6) molecular weight (in 1-3 M urea, 1-2 M methyl- and ethylurea, and 1 M propylurea), as a mixture of hexamers and monomers, the latter with a molecular weight of 250000 (i.e., in 4 M urea), or as a mixture of all three species of duodecamers, hexamers, and monomers, seen in 2 M propylurea. Parallel studies by optical rotation and absorption measurements indicate that there is little or no unfolding of the subunits at urea and alkylurea concentrations where complete dissociation to hexamers and extensive dissociation to monomers can be achieved. Further splitting of the monomers (A subunits) to smaller fragments of one-third to one-quarter of the molecular weight of the monomers (B subunits) is seen in the presence of 7 and 8 M urea (pH 7) and in alkaline urea to propylurea solutions. Analysis of the dissociation data of duodecamers to monomers, based on equations used in studies of the urea and amide dissociation of human hemoglobin A from our laboratory, suggests few urea and alkylurea binding sites at the areas of hexamer contacts in the associated duodecameric form of L. terrestris hemoglobin. This suggests that hydrophobic interactions are not the dominant forces that govern the state of association of L. terrestris hemoglobin relative to polar and ionic interactions. The unfolding effects of the ureas, at concentrations above the dissociation transitions, are closely similar to their effects on other globular proteins, suggesting that hydrophobic interactions play an important role in the maintenance of the folded conformation of the subunits. Use of the Peller-Flory equation, with binding constants based on free energy transfer data of hydrophobic amino acid side chains and denaturation data used in previous denaturation studies, gave a relatively good acount of the observed denaturation midpoints obtained with the various ureas supporting these conclusions.     

A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme.

Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.     

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.     

Polymerization of Bacteriophage T4 Tail Sheath Protein Mutants Truncated at the C-Termini

Gene 18 of bacteriophage T4 encodes the contractile protein of the tail sheath. Previous work has shown that the full-length recombinant gene product (gp) 18 of 658 amino acid residues assembles in Escherichia coli cells into a long polysheath structure. However, the gp18 mutants truncated at the N-termini form insoluble aggregates similar to inclusion bodies. In this study, six plasmid vectors expressing the recombinant gp18 proteins truncated at the C-termini have been constructed. The CDelta58, CDelta129, CDelta152, C[g1]72, CDelta248, and CDelta287 proteins contain 600, 529, 506, 486, 410, and 371 residues of the full-length gp18 molecule, respectively. All the recombinant proteins were soluble and, except for the CDelta287 mutant, were assembled into polysheath-related structures. Electron microscopy of negatively stained purified proteins was performed and the resulting images were analyzed by computing their Fourier transforms. The CDelta58 and CDelta129 mutants, in addition to forming common contracted-type polysheath structures, assembled into thinner filaments that we called "noncontracted polysheaths" (NCP). The CDelta152, CDelta172, and CDelta248 proteins assembled into the NCP type only. Image processing showed that the NCP filaments significantly differ from both extended sheaths of T4 particle and polysheaths. The structure of the NCP filaments might correspond to the transitional helices postulated by Moody (J. Mol. Biol., 1973, 80, 613-636) that appeared during the process of tail contraction. Our results suggest that a short region at the C-terminus of the CDelta129 protein determines the contractile properties of the gp18 molecule. The shortest, the CDelta287 protein, does not assemble into regular structures, thus indicating that a sequence's stretch at the C-end of the CDelta248 mutant might be responsible for polymerization of gp18.     

Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme.

Human erythrocyte nucleoside-diphosphate kinase (NDP kinase) is a hexameric enzyme consisting of two kinds of polypeptide chains, A and B. By random association (A6, A5B...AB5, B6) these polypeptides form isoenzymes differing in their isoelectric point. Chains A and B of NDP kinase were purified by ion-exchange chromatography under denaturing conditions. Upon mixing and renaturation, the isozymic pattern of NDP kinase obtained by conventional methods was restored. Antibodies raised against purified chains showed significant cross-reactivity, both in immunoblot experiments and activity inhibition studies. Sequence determination showed that both chains consisted of 152 amino acid residues corresponding to Mr or 17,143 (chain A) and 17,294 (chain B), respectively. There was high homology between the two sequences (88% identity). The phosphorylation site on the enzyme is located at His-118. Chain A was identical with human Nm23 protein, which has been reported as a potential suppressor protein in tumor metastasis and chain B was identical with Nm23-H2 protein.     

Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.     

Crystal Structures of S120G Mutant and Wild Type of Human Nucleoside Diphosphate Kinase A in Complex with ADP

Nm23 was the first metastasis suppressor gene identified. This gene encodes a NDP kinase that also exhibits other properties like histidine protein kinase and interactions with proteins and DNA. The S120G mutant of NDPK-A has been identified in aggressive neuroblastomas and has been found to reduce the metastasis suppressor effect of Nm23. In order to understand the differences between the wild type and the S120G mutant, we have determined the structure of both mutant and wild type NDPK-A in complex with ADP. Our results reveal that there are no significant changes between the two enzyme versions even in the surroundings of the catalytic histidine that is required for NDP kinase activity. This suggests that the S120G mutation may affect an other protein property than NDP kinase activity.     

Molecular cloning and functional expression of the second mouse nm23/NDP kinase gene, nm23-M2.

A new murine cDNA of nm23/NDP kinase was isolated. A RT-PCR product was obtained from the normal mouse liver mRNA with primers designed for the human nm23-H2 gene. The product was used as a probe to screen a cDNA library from the murine melanoma cell line, B16, and two clones containing the entire open reading frame were obtained. It was predicted that the DNA sequence encoded 152 amino acids which was 98% identical to the nm23-H2 protein. The entire nm23-M1 and -M2 gene-coding regions were translated as fusion proteins with a glutathione S-transferase. These fusion proteins displayed NDP kinase activities.     

Rescue of the neuroblastoma mutant of the human nucleoside diphosphate kinase A/nm23-H1 by the natural osmolyte trimethylamine-N-oxide

The point mutation S120G in human nucleoside diphosphate kinase A, identified in patients with neuroblastoma, causes a protein folding defect. The urea-unfolded protein cannot refold in vitro, and accumulates as a molten globule folding intermediate. We show here that the trimethylamine-N-oxide (TMAO) corrects the folding defect and stimulated subunit association. TMAO also substantially increased the stability to denaturation by urea of both wild-type and S120G mutant. A non-native folding intermediate accumulated in the presence of 4.5-7 M urea and of 2 M TMAO. It was inactive, monomeric, contained some secondary structure but no tertiary structure and displayed a remarkable stability to denaturation.     

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.     

Characterization of the denaturation of human alpha-lactalbumin in urea by molecular dynamics simulations

Molecular dynamics (MD) simulations were used to characterize the non-cooperative denaturation of the molten globule A-state of human alpha-lactalbumin by urea. A solvent of explicit urea and water molecules was used, corresponding to a urea concentration of approximately 6M. Three simulations were performed at temperatures of 293K, 360K and 400K, with lengths of 2 ns, 8 ns and 8 ns respectively. The results of the simulations were compared with experimental data from NMR studies of human alpha-lactalbumin and related peptides. During the simulations, hydrogen bonds were formed from the protein to both urea and water molecules as intra-protein hydrogen bonds were lost. Urea was shown to compete efficiently with water as both a hydrogen bond donor and acceptor. Radial distribution functions of water and urea around hydrophobic side chain atoms showed a significant increase in urea molecules in the solvation shell as the side chains became exposed during denaturation. A considerable portion of the native-like secondary structure persisted throughout the simulations. However, in the simulations at 360K and 400K, there were substantial changes in the packing of aromatic and other hydrophobic side chains in the protein, and many native contacts were lost. The results suggest that during the non-cooperative denaturation of the molten globule, secondary structure elements are stabilized by non-specific, non-native interactions.     

Nm23-H1/NDP kinase folding intermediates and cancer: a hypothesis

The Nm23-H1/nucleoside diphosphate (NDP) kinase A is a metastasis suppressor, besides its enzymatic activity. The mutant S120G has been found in high-grade neuroblastomas. The mutant protein, once denatured in urea, is unable to refold in vitro. A size-exclusion chromatography analysis of the folding/association pathway showed that recombinant wild-type and S120G mutant human Nm23-H1/NDP kinase A unfold and refold passing through a molten globule state while typical hexameric NDP kinases unfold without dissociated species and refold through a native monomeric intermediate. A survey of the recent literature showed that several proteins involved in cancer, and their mutants, are marginally stable, like the wild-type Nm23-H1/NDP kinase A, or are misfolded, like its S120G mutant. We therefore suggest that the low thermodynamic stability and the folding intermediate of the Nm23-H1/NDP kinase A may be necessary for its regulatory properties.     

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.     

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.     

Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1

Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.     

Quaternary Structure of Nucleoside Diphosphate Kinases

Nucleoside (NDP) diphosphate kinases are oligomeric enzymes. Most are hexameric, but somebacterial enzymes are tetrameric. Hexamers and tetramers are constructed by assemblingidentical dimers. The hexameric structure is important for protein stability, as demonstratedby studies with natural mutants (the Killer-of-prune mutant ofDrosophila NDP kinase andthe S120G mutant of the human NDP kinase A in neuroblastomas) and with mutants obtainedby site-directed mutagenesis. It is also essential for enzymic activity. The function of the tetrameric structure is unclear.     

A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.     

Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes.

The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.