Crystallization and preliminary X-ray diffraction analysis of nucleoside diphosphate kinase from Myxococcus xanthus

doi:10.1016/0022-2836(91)90374-F

Journal of Molecular Biology

Volume 220, Issue 1, 5 July 1991, Pages 5-7

https://doi.org/10.1016/0022-2836(91)90374-F Get rights and content

Abstract

Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the γ-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2₁2₁2₁) with $a = 267·1 A ̊, b = 74·0 A ̊ and c = 75·1 A ̊$ for form I and $a = 53·5 A ̊, b = 74·0 A ̊ and c = 75·1 A ̊$ for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0·2 along the a axis. Diffraction data have been recorded to 1·9 Å for form I and to 2·2 Å for form II.

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Cited by (8)

Role of Mg<sup>2+</sup> in nucleoside diphosphate kinase autophosphorylation
1998, Archives of Biochemistry and Biophysics
Nucleoside diphosphate (NDP) kinase is a ubiquitous enzyme that has been described to have regulatory functions. In addition to its classical enzymatic activity, NDP kinases have been characterized as inhibitors of metastasis, as a factor stimulating gene transcription, and as a protein kinase. In this report we show some characteristics of the autophosphorylation of homogeneous NDP kinase and make a comparison with that of other proteins in crude extracts. By using labeled substrates and fluorescence quenching analysis, we prove that Mg²⁺is indeed necessary for the two steps of the ping-pong reaction to take place and present evidence that NTPs or NDPs, when uncomplexed to divalent cations, may not bind the active site in a comparable way to NTP · Mg²⁺and NDP · Mg²⁺. However, even extremely small concentrations of Mg²⁺suffice for maximal autophosphorylation which is obtained with Mg²⁺in the nanomolar range and 100 μM ATP using homogeneous enzyme. Moreover, lower autophosphorylation levels were observed with increasing concentrations of Mg²⁺. The autophosphorylation equilibrium varied from 0.19 to 1.6 upon the inclusion of 10 mM EDTA to produce low Mg²⁺concentrations. Under optimal conditions (low Mg²⁺concentrations and short incubation times) NDP kinase was the only protein phosphorylated in crude extracts fromCandida albicans,indicating that the autophosphorylation properties of the enzyme are very singular.
The in vitro DNA binding properties of NDP kinase are related to its oligomeric state
1997, FEBS Letters
Genetic and biochemical evidences suggest that the enzymatic activity of NDP kinase is necessary but not sufficient for its biological function. While the human NDPK-B binds specifically single-strand polypyrimidines sequences, the hexameric enzyme from Dictyostelium does not. We demonstrated by electrophoretic mobility shift assay and filter binding assay that a dimeric mutant from Dictyostelium binds to an oligodesoxynucleotide while the wild-type does not. These data suggest that the differences in the DNA binding properties of several eucaryotic NDP kinases might be correlated to the differences in the stability of their hexameric structure.
Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture
1996, FEBS Letters
Although a number of nucleoside diphosphate kinases (NDPKs) have been reported to act as inhibitors of metastasis or as a transcription factor in mammals, it is not known whether these functions are linked to their enzymatic activity or how this protein is regulated. In this report, we show that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo ³²P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated on histidine residues, however, only the B isoform appeared to be serine phosphorylated.
Crystal Structure of Myxococcus xanthus Nucleoside Diphosphate Kinase and its Interaction with a Nucleotide Substrate at 2·0 Å Resolution
1993, Journal of Molecular Biology
The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2·0 Å resolution to a crystallographic R-factor of 0·17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in P 4₃2₁2 and I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel βsheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a direct with an extensive interface in which 1092 Å² per monomer is buried. The majority, of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 Å² of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution.
The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg²⁺ interacts with the pyrophosphate of the ADP and via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the β-sheet.
The structures of the nucleotide in crystals grown in the presence or absence of Mg²⁺ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg²⁺. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.
Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes
1991, Gene
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 [γ-³²P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the M_r of the purified E. coli NDP kinase previously reported.
The structure of the Escherichia coli nucleoside diphosphate kinase reveals a new quaternary architecture for this enzyme family
2007, Proteins: Structure, Function and Genetics

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^☆: This work was supported by the Center for Advanced Biotechnology and Medicine and the New Jersey Commission on Science and Technology.

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CommunicationCrystallization and preliminary X-ray diffraction analysis of nucleoside diphosphate kinase from Myxococcus xanthus☆

Abstract

Cell

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Communication
Crystallization and preliminary X-ray diffraction analysis of nucleoside diphosphate kinase from Myxococcus xanthus☆