Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of <i>Mycobacterium tuberculosis</i>

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

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

International audienceMost oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the WT and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding/folding by Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are native-like dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers

Nucléoside diphosphate kinases (repliement et stabilité)

Les Nucléoside Diphosphate kinases (NDPK) sont des enzymes responsables de la synthèse des nucléosides triphosphates. Hexamériques chez les eucaryotes, leur structure stabilise les monomères. Chez l'homme, l'isoforme A est codé par le gène Nm23 lequel est également un gène suppresseur de métastases. La mutation naturelle S120G déstabilise les monomères qui perdent leur capacité à se renaturer correctement et restent in vitro sous forme d'intermédiaires de repliement de type molten-globule. (1) Nous avons montré que l'ATP corrige le défaut de repliement du mutant S120G en phosphorylant son histidine catalytique. Ce mécanisme physiologiquement pertinent explique pourquoi la protéine mutée est fonctionnelle dans les cellules cancéreuses. Le chaperon chimique triméthylamine-N-oxyde corrige également le défaut de repliement du mutant S120G, mais par un mécanisme différent de celui de la phosphorylation. Ces deux mécanismes ont un effet additif. Enfin, l'introduction d'une deuxième mutation "suppresseur intra-génique" corrige partiellement le défaut de repliement. (2) Nous avons également montré que le mutant S120G peut adopter une conformation alternative aberrante et former des fibres amyloides en feuillets b. Plusieurs modèles corrèlent la stabilité des enzymes oligomériques à l'aire d'interface enfouie. Plus l'interface est grande, plus le complexe est thermostable. La NDPK de M. tuberculosis est hexamérique et présente une interface de 30 % plus faible que celle d'autres NDPK (celle de D. discoideum par exemple) pour une stabilité thermique plus élevée. (3) La stabilité de cette NDPK a été étudiée pour comprendre ce paradoxe au niveau structural.Nucleoside Diphosphate kinases (NDPK) are enzymes responsible of nucleoside triphosphate synthesis. Hexameric in eukaryotes, their quaternary structure stabilises monomers. The human isoforme A is encoded by the Nm23 gene which is also known to be a metastasis suppressor. The S120G natural mutation destabilises monomers which become unable to refold correctly and stay in vitro under a molten-globule like intermediate conformation. (1) We have shown that ATP corrects the S120G mutant folding defect by phosphorylating its catalytic histidine. The refolding mechanism by phosphorylation is pertinent physiologically and explains why the mutated protein is functional in tumoral cells. The presence of the chemical chaperone trimethylamine-N-oxyde corrects the folding defect of the S120G mutant but by a mechanism different of that of phosphorylation. Both mechanisms have an additive effect. Introduction of a second site suppressor mutation partially corrects the S120G mutant folding defect. (2) Moreover, we have shown that the S120G mutant can adopt an alternative aberrant conformation and form amyloid fibres rich in b sheets. Several models correlate the stability of oligomeric enzymes to the interface area. Larger is the interface, more thermostable is the complex. The M. tuberculosis NDPK is hexameric and presents 30 % less of interface, as compared to the others NDPK (such as Dictyostelium discoideum, for example) the thermostability of which is higher. (3) We have studied the NDPK stability in order to understand such a paradox at the structural level.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Remodeling of the Binding Site of Nucleoside Diphosphate Kinase Revealed by X-ray Structure and H/D Exchange

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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

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

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

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