Giraud et al (2006) IJBBM - Crystal structures of S120G mutant and wild type of human nucleoside diphosphate kinase A in complex with ADP (original) (raw)
Related papers
2006
Nm23 was the first metastasis suppressor gene 7 identified. This gene encodes a NDP kinase that also exhibits 8 other properties like histidine protein kinase and interactions 9 with proteins and DNA. The S120G mutant of NDPK-A has 10 been identified in aggressive neuroblastomas and has been 11 found to reduce the metastasis suppressor effect of Nm23. In 12 order to understand the differences between the wild type and 13 the S120G mutant, we have determined the structure of both 14 mutant and wild type NDPK-A in complex with ADP. Our 15 results reveal that there are no significant changes between 16 the two enzyme versions even in the surroundings of the 17 catalytic histidine that is required for NDP kinase activity. 18 This suggests that the S120G mutation may affect an other 19 protein property than NDP kinase activity. 20
Clinical & experimental metastasis, 2002
Tumor metastasis is responsible for a high degree of mortality in cancer patients. One of the genes involved in tumor metastasis is NM23. At present, eight human isoforms, transcribed from different NM23 genes, have been detected. The gene products have been identified as nucleoside diphosphate kinases (NDPKs), most of which catalyse the transfer of the gamma-phosphate of a (deoxy)nucleoside triphosphate to a (deoxy)nucleoside diphosphate. However, the function of NDPK isoforms involved in tumor metastasis cannot be explained on the basis of their phosphotransferase activity alone. At present, several other properties, like transcriptional regulation and protein kinase activity, have been assigned to these proteins. Moreover, it has also been shown that NDPKs interact with several other proteins, and binding partners of NDPKs are identified at an increasing rate. Accumulating evidence indicates that protein-protein interactions modulate the molecular action of NDPKs. In this review ...
Overexpression of nucleoside diphosphate kinase (nm23) in solid tumours
European Journal of Cancer and Clinical Oncology, 1991
The product of the nm23-Hl gene, reported to be related to the metastatic potential of tumour cells, was recently identified as the nucleoside diphosphate (NDP) kinase A (Gilles et al., 1991, J Biol Chem, 266, 8784-8789). An analysis of the enzyme by activity measurement and immunological techniques using polyclonal antibodies raised against the NDP kinase A purified from human erythrocytes, was performed on 39 human tissue specimens. Markedly increased activity and higher level of the protein were observed in extracts of solid tumours as compared to the corresponding normal tissues (P < 0.01). An intense immunolabelling of tumoral cells was observed in sections of the malignant tumours and of some but not all benign neoplasia. The staining is observed in noninvasive and invasive ductal breast carcinomas with or without lymph node involvement as well as in colon and cervix carcinomas and in a case of metastatic melanoma. Therefore, NDP kinase A level is increased in neoplastic tissues but no correlation with metastatic potential could be demonstrated.
The Crystal Structure of Human Nucleoside Diphosphate Kinase, NM23-H2
Journal of Molecular Biology, 1995
The 2.8 Å resolution X-ray structure of NM23-H2 has been determined by molecular replacement using the structure of Myxococcus xanthus nucleoside Engineering, MRC Centre Hills Road, Cambridge diphosphate (NDP) kinase. NM23-H2 is a human NDP kinase. The enzyme catalyses phosphoryl transfer, binds DNA, and can activate the transcription CB2 2QH, UK of the c-myc oncogene in vitro. NM23 has also been reported to be a 2 Molecular Biology Research suppressor of metastasis in some types of tumours. Whereas the M. xanthus Section, Lederle Laboratories NDP kinase is a tetramer, NM23-H2 is a hexamer. The fold of NM23-H2 is Pearl River, New York 10965 identical to the fold of other NDP kinases. Two antiparallel helices joined by USA a turn form one edge of the nucleotide binding cleft. This region moves in a hinge-like fashion in response to substrate binding and crystal packing forces. Additional differences in conformation among the NDP kinases are principally in regions involved in protein-protein contacts within the oligomers. The only protein-protein interaction conserved among all NDP kinases is a dimeric interaction. Several mutations of NM23-H2 have been detected in tumour tissues. These mutations do not involve residues interacting with the substrates, and probably destabilise the enzyme without directly affecting the catalytic activity. Low level phosphorylation of serines has been reported for NM23 both in vitro and in vivo. The structure of the hexamer indicates that two serine residues that have been reported as being phosphorylated, Ser44 and Ser122, are on the surface of the hexamer, and are likely to be phosphorylated by exogenous kinases. In contrast, Ser120 is buried, and is most likely phosphorylated by a direct transfer from the phosphohistidine intermediate of the reaction mechanism.
Journal of Molecular Biology, 2003
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 2.0 Å resolution of the variant NDPK-A in complex with ADP, Ca 2þ 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 b and g-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.
Biochemical Journal, 2007
Human nucleoside diphosphate (NDP) kinase A is a 'housekeeping' 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 ureadenatured 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 -adenylylimido-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.
Journal of Biological Chemistry, 1997
The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.
Experimental Cell Research, 1999
The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), encoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homoor heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific functions. The nm23-H1 gene is implicated in tumor progression and metastasis, and the nm23-H2 gene product is a transcription factor for c-myc. To determine if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluorescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B was shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates.