X-ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase



Nucleoside Diphosphate (NDP) kinases transfer the γ-phosphate of a nucleoside or deoxynucleoside triphosphate, usually ATP, to a nucleoside or deoxynucleoside diphosphate, yielding the substrates of RNA and DNA synthesis. The genes, present in almost all organisms, code for polypeptide chains of about 150 residues with very similar sequences and folds. Nevertheless, the quaternary structure (reviewed in Lascu et al.1) is not conserved: most NDP kinases are hexamers, but tetramers are found in some bacteria. The tetramer is illustrated by the X-ray structure of the Myxococcus xanthus enzyme,2 the hexamer, by those of Dictyostelium, Drosophila, and several human and bovine isoforms (reviewed in Janin et al.3). The eukaryotic gene products are 10–12 residues longer at the C-terminus than in Myxococcus. Because major intersubunit contacts implicate these residues in the hexamer, short-chain bacterial NDP kinases were assumed to be tetramers like Myxococcus. We present here the 2.6 Å X-ray structure of the enzyme from Mycobacterium tuberculosis, which has an even shorter polypeptide chain, and show that it forms a very stable hexamer despite the missing interactions.

Crystallization and Structure Determination.

Cloning of the M. tuberculosis gene, expression in E. coli, purification, and biochemical characterization of the recombinant protein will be described elsewhere (Lascu et al., in preparation). Crystals of the recombinant protein appeared within 1 week in hanging drops containing 8 mg/mL protein, 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 100 mM Na citrate, 20% iso-propanol, and 20% PEG 4000. They belong to the hexagonal space group P212121 with unit cell a = 77.0 Å, b = 98.8 Å, and c = 127.35 Å. The asymmetric unit contains a hexamer.

X-ray diffraction data were collected from a single crystal at 20°C on a Rigaku X-ray generator (λ = 1.542 Å) with a MARresearch detector. The data, evaluated with DENZO and SCALEPACK,4 were 99.5% complete, with I/σ = 11.7 and a redundancy of 5.7 at 2.6 Å resolution. Molecular replacement was performed with AMoRe5 by using the human NDP kinase B monomer (entry 1NUE6) as a search model. Refinement by CNS7 yielded a model with Rcryst = 20.5% and Rfree = 24.6% for all data at 2.6 Å resolution, and good geometry. The model contains all 810 residues of the hexamer (residue 1 is missing) and 107 solvent atoms. Coordinates have been deposited at the Protein Data Bank (entry 1K44).

The M. tuberculosis subunit.

M. tuberculosis and human B NDP kinases have 45% sequence identity over 135 common residues. A single residue is inserted at position 97 of the bacterial sequence. Figure 1 shows that the subunit folds are very similar, with a root-mean-square distance (RMSD) of 0.94 Å between all equivalent Cα positions. The nucleotide-binding site and the active site are essentially unchanged. Nevertheless, main-chain movements of 2–5 Å are seen at residues 97–99 and at the C-terminus. Residues 97–99, which include the insertion site, belong to the Kpn loop, named so in reference to a Drosophila mutant, which substitutes a proline residue in this loop.8 The Kpn loop, located near the threefold axis of the hexamer, is heavily involved in subunit contacts.9 The carboxylate of C-terminal Ala136 is firmly held in place by a salt bridge with Arg4, effectively closing the polypeptide chain on itself. In the human protein, the chain is 15 residues longer, and the C-terminal Glu152 interacts with another subunit in the hexamer.6

Figure 1.

The subunit of M. tuberculosis NDP kinase (blue ribbon) is superimposed on human NDP kinase B (also called Nm23-H2, red tube), which is longer by 15 residues at the C-terminus. Arg4, linked by a salt bridge to the terminal carboxylate of Ala136, and Ala 97 inserted in the Kpn loop are drawn in full bonds. A GDP molecule bound to the human B subunit6 locates the substrate-binding site.

Subunit Interactions.

Despite its divergent sequence in the Kpn loop and shorter C-terminus, M. tuberculosis NDP kinase is a hexamer like its eukaryotic homologs. It has excellent (noncrystallographic) D3 symmetry, the RMSD between equivalent Cα positions in all six subunits being 0.2–0.4 Å. Dimer contacts along the twofold axes bury 590 Å2 per subunit. This is similar to the 640 Å2 buried in Dictyostelium NDP kinase, but represents only 60% of the area buried in the human or Drosophila homologs.9 The trimer contacts around the threefold axis are also less extensive than in the eukaryotic proteins, burying 1140 Å2 per subunit instead of 1700 Å2 in Drosophila. In either case, the eukaryotic C-terminal extension makes up for the difference. The dimer interfaces contain three hydrogen bonds, the trimer interfaces, five per subunit, fewer than in the eukaryotic proteins, where the C-terminal extension makes additional polar interactions.9 On the other hand, a salt bridge is formed by Arg80 and Asp93 within trimers. This interaction is specific to M. tuberculosis, neutral side-chains occupying positions equivalent to 80 and 93 in most other NDP kinase sequences.


M. tuberculosis NDP kinase is quite thermostable, remaining 100% active up to 75°C when heated at a rate of 1°C per minute. Above that temperature, inactivation occurs sharply and is complete at 82°C (data not shown). Thus, a stable hexamer assembly is maintained despite the shorter sequence, smaller interfaces, and fewer polar interactions between subunits compared with other hexameric NDP kinases. The origin of this stability in currently under study.


We thank Ms. Françoise Borne for cloning the M. tuberculosis gene in an expression vector.