Abstract
Family I soluble inorganic pyrophosphatases (PPases; EC 3.6.1.1) are enzymes essential for all organisms. They hydrolyze inorganic pyrophosphate, thus providing the driving force for numerous biosynthetic reactions. Soluble PPases retain enzymatic activity only in multimeric forms. PPases from various organisms are extensively studied by X-ray crystallography but until now there was no information on their structure and dynamics in solution. Hexameric 110 kDa (6 × 18.3 kDa) PPase from Mycobacterium tuberculosis (Mt-PPase) is a promising target for the rational design of potential anti-tuberculosis agents. In order to use NMR techniques in functional studies of Mt-PPase and rational design of the inhibitors for this enzyme, it is necessary to have information on the backbone 1H, 13C and 15N resonance assignments. Samples of Mt-PPase enriched with 99% of 13C and 15N isotopes, and 95% of 2H were obtained using recombinant protein expression in an isotopically-labeled medium and effective heat-shock protocol for the deuterium-to-hydrogen exchange of the amide groups. Backbone resonance assignment was achieved for more than 95% of the residues. It was found that the secondary structure of Mt-PPase in solution corresponds well to the crystal structure of this protein. Protein backbone dynamics were studied using 15N NMR relaxation experiments. Determined resonance assignments and dynamic properties provide the basis for the subsequent structure-based design of novel inhibitors of Mt-PPase—potential anti-tuberculosis drugs.
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Abbreviations
- PPase:
-
Pyrophosphatase
- Mt-PPase:
-
PPase from Mycobacterium tuberculosis
- Ec-PPase:
-
PPase from Escherichia coli
- Pi :
-
Inorganic phosphate
- PPi :
-
Inorganic pyrophosphate
- τc :
-
Correlation time of protein tumbling
- τe :
-
Effective correlation time of internal motion
- R1 :
-
Longitudinal or spin–lattice relaxation rate
- R2 :
-
Transverse or spin–spin relaxation rate
- Rex :
-
Conformational exchange contribution to R2
- S2 :
-
Order parameter reflecting the amplitude of ps–ns bond vector dynamics
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Acknowledgements
This work was supported by the Russian Science Foundation (Grant 19-14-00115). The authors are grateful to Moscow State University (Russia) for the opportunity to use the NMR facilities and MALDI mass-spectrometer. The authors are also grateful to Svetlana A. Kurilova (A.N. Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russia) for providing pET23a vector with Mt-PPase gene, and to Marina V. Serebryakova for performing protein mass-spectrometry analysis. The authors acknowledge the support from the Russian Government Program of Competitive Growth of Kazan Federal University among the world’s leading academic centers and the NMR equipment at the KFU Center of shared facilities.
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Romanov, R.S., Mariasina, S.S., Efimov, S.V. et al. Backbone resonance assignment and dynamics of 110 kDa hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis. Biomol NMR Assign 14, 281–287 (2020). https://doi.org/10.1007/s12104-020-09962-7
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DOI: https://doi.org/10.1007/s12104-020-09962-7