A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes
| Autor: | Ibolya Leveles, Ábris Bendes, Beáta G. Vértessy, Balázs Jójárt, Judit Tóth, Anna Lopata, Béla Viskolcz |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Molecular model In silico Molecular Dynamics Simulation 010402 general chemistry 01 natural sciences Biochemistry Pyrophosphate 03 medical and health sciences chemistry.chemical_compound Molecular dynamics Bacterial Proteins Catalytic Domain Nucleotide Pyrophosphatases Molecular Biology chemistry.chemical_classification biology 01.04. Kémiai tudományok Active site Mycobacterium tuberculosis Cell Biology 0104 chemical sciences Amino acid 030104 developmental biology Enzyme chemistry Enzymology biology.protein |
| DOI: | 10.1074/jbc.M116.734012 |
| Popis: | 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, the RAMD approach 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 shuttlingof the nucleotides between the binding pocket and the solvent. |
| Databáze: | OpenAIRE |
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