A hidden active site in the potential drug target mycobacterium tuberculosis dUTPase is accessible through small amplitude protein conformational changes

Anna Lopata, Ibolya Leveles, Ábris Ádám Bendes, Béla Viskolcz, Beáta G. Vértessy, Balázs Jójárt, J. Tóth

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, theRAMDapproach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttling of the nucleotides between the binding pocket and the solvent.

Original languageEnglish
Pages (from-to)26320-26331
Number of pages12
JournalJournal of Biological Chemistry
Volume291
Issue number51
DOIs
Publication statusPublished - Dec 16 2016

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Mycobacterium tuberculosis
Catalytic Domain
Nucleotides
Pharmaceutical Preparations
Molecular Dynamics Simulation
Proteins
Shielding
Molecular dynamics
DNA
Enzymes
Mycobacterium
Ports and harbors
Metabolism
Computer Simulation
Conformations
Hydrolysis
dUTP pyrophosphatase
Ligands
Amino Acids
Kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A hidden active site in the potential drug target mycobacterium tuberculosis dUTPase is accessible through small amplitude protein conformational changes. / Lopata, Anna; Leveles, Ibolya; Bendes, Ábris Ádám; Viskolcz, Béla; Vértessy, Beáta G.; Jójárt, Balázs; Tóth, J.

In: Journal of Biological Chemistry, Vol. 291, No. 51, 16.12.2016, p. 26320-26331.

Research output: Contribution to journalArticle

Lopata, Anna ; Leveles, Ibolya ; Bendes, Ábris Ádám ; Viskolcz, Béla ; Vértessy, Beáta G. ; Jójárt, Balázs ; Tóth, J. / A hidden active site in the potential drug target mycobacterium tuberculosis dUTPase is accessible through small amplitude protein conformational changes. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 51. pp. 26320-26331.
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