Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins.
Ontology highlight
ABSTRACT:
SUBMITTER: Islam K
PROVIDER: S-EPMC1152934 | biostudies-other | 1985 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.
| S-EPMC1152935 | biostudies-other
Project description:To be effective as antiviral agent, AZT (3'-azido-3'-deoxythymidine) must be converted to a triphosphate derivative by cellular kinases. The conversion is inefficient and, to understand why AZT diphosphate is a poor substrate of nucleoside diphosphate (NDP) kinase, we determined a 2.3-A x-ray structure of a complex with the N119A point mutant of Dictyostelium NDP kinase. It shows that the analog binds at the same site and, except for the sugar ring pucker which is different, binds in the same way as the natural substrate thymidine diphosphate. However, the azido group that replaces the 3'OH of the deoxyribose in AZT displaces a lysine side chain involved in catalysis. Moreover, it is unable to make an internal hydrogen bond to the oxygen bridging the beta- and gamma-phosphate, which plays an important part in phosphate transfer.
| S-EPMC23778 | biostudies-literature
Project description:The analysis of the Acanthamoeba polyphaga mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 A to 1.5 A. Our results suggest that the mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.
| S-EPMC2704768 | biostudies-literature
Project description:The radial spokes are required for Ca(2+)-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca(2+)-independent manner, whereas IQ2 and IQ3 show Ca(2+)-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca(2+). This Ca(2+)-responsive enzyme, which accounts for approximately 45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca(2+)-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.
| S-EPMC491844 | biostudies-literature
Project description:The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had approximately 20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance.
| S-EPMC326647 | biostudies-literature
Project description:Nephronophthisis (NPH) is an autosomal recessive renal disease leading to kidney failure in children and young adults. The protein products of the corresponding genes (NPHPs) are localized in primary cilia or their appendages. Only about 70% of affected individuals have a mutation in one of 100 renal ciliopathy genes, and no unifying pathogenic mechanism has been identified. Recently, some NPHPs, including NIMA-related kinase 8 (NEK8) and centrosomal protein 164 (CEP164), have been found to act in the DNA-damage response pathway and to contribute to genome stability. Here, we show that NME/NM23 nucleoside-diphosphate kinase 3 (NME3) that has recently been found to facilitate DNA-repair mechanisms binds to several NPHPs, including NEK8, CEP164, and ankyrin repeat and sterile α motif domain-containing 6 (ANKS6). Depletion of nme3 in zebrafish and Xenopus resulted in typical ciliopathy-associated phenotypes, such as renal malformations and left-right asymmetry defects. We further found that endogenous NME3 localizes to the basal body and that it associates also with centrosomal proteins, such as NEK6, which regulates cell cycle arrest after DNA damage. The ciliopathy-typical manifestations of NME3 depletion in two vertebrate in vivo models, the biochemical association of NME3 with validated NPHPs, and its localization to the basal body reveal a role for NME3 in ciliary function. We conclude that mutations in the NME3 gene may aggravate the ciliopathy phenotypes observed in humans.
| S-EPMC6166740 | biostudies-literature
Project description:Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.
| S-EPMC3589492 | biostudies-literature
Project description:Leishmania amazonensis was found to release nucleoside diphosphate kinase (NdK)-a stable enzyme capable of decreasing extracellular ATP. The release of this enzyme from Leishmania results in its progressive accumulation extracellularly as they replicate, peaking at the stationary phase in vitro. The released NdK is immunoprecipitable and constitutes approximately 40% of its total activities and proteins. The retention of a known cytosolic protein by wild type cells and a fluorescent protein by DsRed transfectants at stationary phase, which release NdK, indicates that this is a spontaneous event, independent of inadvertent cytolysis. Recombinant products of Leishmania NdK prepared were enzymatically and immunologically active. Both recombinant and native Leishmania NdK utilized ATP to produce expected nucleoside triphosphates in the presence of nucleoside diphosphates in excess. Both native and recombinant Leishmania NdK were also found to prevent ATP-induced cytolysis of J774 macrophages in vitro, as determined by assays for lactate dehydrogenase release from these cells and for their mitochondrial membrane potential changes. The results obtained thus suggest that Leishmania NdK not only serves its normal house-keeping and other important functions true to all cells, but also prevents ATP-mediated lysis of macrophages, thereby preserving the integrity of the host cells to the benefit of the parasite.
| S-EPMC2277215 | biostudies-literature
Project description:Co-enzyme A is the essential cellular carrier of the acetyl group, largely used in fatty acid synthesis and lysine post-translational modifications of proteins. Following the analysis of the cellular CoA-binding proteome, we identified Nme1/2 as major abundant cytoplasmic CoA-binding proteins. We report a novel mechanism that controls fatty acids synthesis involving ATP-dependent acetyl-CoA binding by Nucleoside Diphosphate Kinases 1 and 2 (Nme1/2).
2023-08-01 | PXD029159 | Pride
Project description:Nucleoside diphosphate kinase (NDPK/Nm23), responsible for intracellular di- and triphosphonucleoside homeostasis, plays multiple roles in cellular energetics, signaling, proliferation, differentiation and tumor invasion. The only human NDPK with a mitochondrial targeting sequence is NDPK-D, the NME4 gene product, which is a peripheral protein of mitochondrial membranes. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. Surface plasmon resonance analysis of the interaction using recombinant NDPK-D and model liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin. Mutation of the central arginine (Arg-90) in a surface-exposed basic RRK motif unique to NDPK-D strongly reduced interaction with anionic phospholipids. Due to its symmetrical hexameric structure, NDPK-D was able to cross-link anionic phospholipid-containing liposomes, suggesting that NDPK-D could promote intermembrane contacts. Latency assays with isolated mitochondria and antibody binding to mitoplasts indicated a dual orientation for NDPK-D. In HeLa cells, stable expression of wild type but not of the R90D mutant led to membrane-bound enzyme in vivo. Respiration was significantly stimulated by the NDPK substrate TDP in mitochondria containing wild-type NDPK-D, but not in those expressing the R90D mutant, which is catalytically equally active. This indicates local ADP regeneration in the mitochondrial intermembrane space and a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on its membrane-bound state.
| S-EPMC3258864 | biostudies-literature