Buy article online - an online subscription or single-article purchase is required to access this article.
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.
Supporting information
 | Joint Photographic Experts Group (JPEG) image https://doi.org/10.1107/S2053230X13034134/hv5248sup1.jpg
Figure S1. Thermal denaturation. The R80N mutant decreased by 8 degrees Celcius and behaves more similarly to the wild-type protein than to the D93N mutation, for which a dramatic decrease of 28 degrees Celcius was observed. The temperature dependence of the excess molar heat capacity of wild-type Mt-NDPK (full circles), the R80N mutant (open squares) and the D93N mutant (full squares) is shown. Each differential scanning calorimetry curve displays a single calorimetric peak. The protein concentration was 0.2-0.3 mg ml-1. |
| Joint Photographic Experts Group (JPEG) image https://doi.org/10.1107/S2053230X13034134/hv5248sup2.jpg
Figure S2. Denaturation/renaturation by urea/GuHCl of wild-type and R80N mutant Mt-NDPK. The unfolding and the loss of quaternary structure are indicated by the excess heat and the enzymatic activity, respectively. Unfolding (full squares) and subsequent refolding (open squares) were monitored by following the intrinsic fluorescence of Mt-NDPK (A in urea, B in GuHCl) and R80N mutant (C in urea, D in GuHCl). The residual enzymatic activity for the unfolding is shown by open circles. The protein concentration was 10 mg ml-1. The measurements are normalized to the maxima; fn is the fraction of native protein. |
 | Portable Document Format (PDF) file https://doi.org/10.1107/S2053230X13034134/hv5248sup3.pdf
Figure S3. Binding scheme of citrate in chain A of D80N mutant Mt-NDPK. The figure was drawn with LigPlot+ (Laskowski and Swindells, 2011). |
PDB reference: nucleoside diphosphate kinase, R80N mutant, 4ane
Subscribe to Acta Crystallographica Section F: Structural Biology Communications
The full text of this article is available to subscribers to the journal.
If you have already registered and are using a computer listed in your registration details, please email
support@iucr.org for assistance.
journal menu
IYCr crystallization series
Subscribe to Acta Crystallographica Section F: Structural Biology Communications
