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Structure of Mycobacterium tuberculosis nucleoside diphosphate kinase R80N mutant in complex with citrate.

ABSTRACT: The crystal structure of the wild-type nucleoside diphosphate kinase from Mycobacterium tuberculosis at 2.6?Å resolution revealed that the intersubunit salt bridge Arg80-Asp93 contributes to the thermal stability of the hexamer (Tm = 76°C). On mutating Asp93 to Asn to break the salt bridge, the thermal stability dramatically decreased by 27.6°C. Here, on mutating Arg80 to Asn, the thermal stability also significantly decreased by 8.0°C. In the X-ray structure of the R80N mutant solved at 1.9?Å resolution the salt bridge was replaced by intersubunit hydrogen bonds that contribute to the thermal stability of the hexamer. A citrate anion from the crystallization buffer was bound at the bottom of the nucleotide-binding site via electrostatic and hydrogen-bonding interactions with six conserved residues involved in nucleotide binding. Structural analysis shows that the citrate is present at the location of the nucleotide phosphate groups.

SUBMITTER: Georgescauld F

PROVIDER: S-EPMC3943099 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Structure of Mycobacterium tuberculosis nucleoside diphosphate kinase R80N mutant in complex with citrate.

Georgescauld Florian F Moynié Lucile L Habersetzer Johann J Dautant Alain A

Acta crystallographica. Section F, Structural biology communications 20131224 Pt 1

The crystal structure of the wild-type nucleoside diphosphate kinase from Mycobacterium tuberculosis at 2.6 Å resolution revealed that the intersubunit salt bridge Arg80-Asp93 contributes to the thermal stability of the hexamer (Tm = 76°C). On mutating Asp93 to Asn to break the salt bridge, the thermal stability dramatically decreased by 27.6°C. Here, on mutating Arg80 to Asn, the thermal stability also significantly decreased by 8.0°C. In the X-ray structure of the R80N mutant solved at 1.9 Å r ...[more]

PMID: 24419614

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Project description:Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

Dataset Information

Structure of Mycobacterium tuberculosis nucleoside diphosphate kinase R80N mutant in complex with citrate.

Publications

Structure of Mycobacterium tuberculosis nucleoside diphosphate kinase R80N mutant in complex with citrate.

Similar Datasets

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