Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of <em>Mycobacterium tuberculosis</em>

<div><p>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 <i>Mycobacterium tuberculosis</i> (<i>Mt</i>) this tail is missing. The quaternary structure of <i>Mt</i>-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg⁸⁰-Asp⁹³ 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.</p> </div

Thermostability of wild type <i>Mt</i>-NDPK and D93N mutant.

<p>The temperature dependence of excess molar heat capacity of the wild-type <i>Mt</i>-NDPK (in red) and D93N mutant (in blue). Each DSC curve displays a single calorimetric peak. The protein concentration was 0.2–0.3 mg/mL.</p

X-Ray data processing and refinement statistics.

a<p>Statistics for the highest resolution bin are shown in parentheses. <i>^b</i>R_sym were calculated by , where <i>h</i> is the index for unique reflections and <i>j</i> is the index for symmetry redundant reflections. <i>I_h</i> is the mean weighted intensity after rejection of outliers. <i>^c</i>R_work and R_free were calculated by Σ||F_observed|−<i>k</i>|F_calculated||/Σ|F_observed|. R_free was calculated using 5% random data omitted from refinement. <i>^d</i>Percentage of Ramachandran outliers and favored.</p

Crystal structures of wild-type <i>Mt</i>-NDPK and D93N mutant.

<p>View along the 3-fold axis of the hexamer of a “trimer” of the wild-type <i>Mt</i>-NDPK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057867#pone.0057867-Chen1" target="_blank">[13]</a> (<b>A</b>). The intersubunit salt bridge found in the wild-type <i>Mt</i>-NDPK (pdb id: 1k44) (<b>B</b>) was clearly broken in the D93N mutant (pdb id: 2and) (<b>C</b>). The side-chain atoms of residues Arg⁹⁰, Gln⁹⁶ and Asp⁹³ or Asn⁹³ and the main-chain atoms of Leu¹⁰⁹ were drawn as sticks. Arg^80# marked the arginine from the neighboring subunit. Non bonded interactions were drawn as broken lines.</p

Denaturation/renaturation by urea/GuHCl.

<p>Unfolding (red circles) and subsequent refolding (blue circles) were monitored by following the intrinsic fluorescence of <i>Mt</i>-NDPK (<b>A</b> in urea, <b>C</b> in GuHCl) and D93N mutant (<b>B</b> in urea, <b>D</b> in GuHCl). The residual enzymatic activity for the unfolding was shown by red squares. The protein concentration was 10 µg/mL. The measurements are normalized to the maxima; f_n is the fraction of native protein.</p

Sequence alignment of NDPKs whose structure has been solved.

<p>Sequence alignment was performed using ClustalW and mapped onto the secondary structure elements of <i>Mt</i>-NDPK, which derived from the crystal structure (PDB id 1k44) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057867#pone.0057867-Chen1" target="_blank">[13]</a>, by ESPript (<a href="http://espript.ibcp.fr/ESPript/ESPript/" target="_blank">http://espript.ibcp.fr/ESPript/ESPript/</a>). The Kpn loop was named after the killer of prune (Kpn) mutation of Drosophila. Among the fully conserved residues indicated on red background, the activesite residues are denoted with a blue star. Triangles indicate Arg80 and Asp93 which form the salt bridge discussed in this paper. The quaternary structure and the pdb code are indicated at the end of the sequences. The enzymes of the first group from <i>M. tuberculosis</i> to <i>B. halodenitrificans</i> are hexameric, while the second tetrameric or dimeric (<i>H. sp</i>. 593).</p

Thermal unfolding of wild-type <i>Mt</i>-NDPK and D93N mutant monitored by CD at 222 nm.

<p>The experiments were performed with wild type <i>Mt</i>-NDPK (<b>A</b>) and D93N mutant (<b>B</b>) in the absence of added salt (black) and in the presence of 0.15 M sodium chloride (blue) or 0.15 M GuHCl (red). The reduction of the absolute molar mean-residue ellipticity at 222 nm (θ_MRE) was a measure of the loss of secondary structure.</p

Determination of the <i>Mt</i>-NDPK stability at the monomeric state by a double dilution experiment.

<p>The <i>Mt</i>-NDPK was first unfolded in 8 M urea or 5 M GuHCl, then refolded for 10 sec by 10-fold dilution in buffer, which is sufficient to allow subunit folding but not for subunit association. Unfolding curves of <i>Mt</i>-NDPK at the monomeric state were obtained by further incubating the proteins for 16 h at 25° at the concentration of denaturant as indicated. The final protein concentration was 11 µg/mL. Circles indicate experimental data in GuHCl, while squares refer to data in urea. Red and blue symbols refer to denaturation and renaturation, respectively. f_n is the fraction of native protein. The ΔGH₂0 calculated was 4.7±0.3 kcal/mol in GuHCl and 5.0±0.5 kcal/mol in urea.</p

GuHCl and other salts promote association of urea-dissociated D93N mutant of <i>Mt-</i>NDPK.

<p>100 µl of protein at 10 µg/mL was incubated for 16 h in 1.5 M urea, in the absence or the presence of salt. (<b>A</b>) Size-exclusion chromatographic analysis, with the D93N mutant in 1.5 M urea (blue), in 1.5 M GuHCl (red) and in 1.5 M urea plus 1.0 M GuHCl (orange) injected into a Superdex 75 10/300 column and the intrinsic protein fluorescence was recorded. The elution profile of <i>Mt</i>-NDPK incubated with 1.5 M GuHCl (empty circles) is shown for comparison. Expected positions for folded monomer (M, 14.5 kDa) and hexamer (H, 87.0 kDa) are indicated. (<b>B</b>) Measurement of residual activity of the D93N mutant, at 10 µg/ml was incubated for 16 h at 25°C in the presence of 1.5 M of urea plus monovalent (squares) and divalent (circles) salts: GuHCl (orange), NH₄Cl (cyan), NaCl (green), MgCl₂ (yellow) or CaCl₂ (red). The enzymatic activity was measured with the standard assay. The lines do not represent theoretical models but were drawn to help the reader.</p

Structural properties of the hexameric NDPKs discussed in the text.

<p>The rmsd were calculated versus the wild-type <i>Mt</i>-NDPK structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057867#pone.0057867-Chen1" target="_blank">[13]</a>. Buried surface area (bsa) are calculated by subunit. The bsa is expected to contribute about 20 cal/mol for each Ų of hydrophobic contact. Nr. a. a., numbers of residues in protein.</p