Backbone resonance assignment and dynamics of 110 kDa hexameric inorganic pyrophosphatase from Mycobacterium tuberculosi
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Backbone resonance assignment and dynamics of 110 kDa hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis Roman S. Romanov1 · Sofia S. Mariasina1 · Sergey V. Efimov2 · Vladimir V. Klochkov2 · Elena V. Rodina1 · Vladimir I. Polshakov3 Received: 6 May 2020 / Accepted: 17 June 2020 © Springer Nature B.V. 2020
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 isotopicallylabeled 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. Keywords Inorganic pyrophosphatase · Mycobacterium tuberculosis · Protein NMR · Resonance assignment · Deuteration 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
* Vladimir I. Polshakov [email protected] 1
Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia 119991
2
NMR Laboratory, Institute of Physics, Kazan Federal University, 18 Kremlevskaya St., Kazan, Russia 420008
3
Center for Magnetic Tomography and Spectroscopy, Faculty of Fundamental Medicine, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia
Rex Conformational exchange contribution to R2 S2 Order parameter reflecting the amplitude of ps–ns bond vector dynamics
Biological context Inorganic pyrophosphatases (PPases; EC 3.6.1.1) are metaldependent enzymes catalyzing the hydrolysis of inorganic pyrophosphate PPi to phosphate Pi. Soluble PPases
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