A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes.
  • Metaadatok
Tartalom: http://real.mtak.hu/75161/
Archívum: MTA Könyvtár
Gyűjtemény: Status = Published

Type = Article
Cím:
A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes.
Létrehozó:
Lopata, Anna
Leveles, Ibolya
Bendes, Ábris Ádåm
Viskolcz, BĂŠla
G. VĂŠrtessy, BeĂĄta
JĂłjĂĄrt, BalĂĄzs
TĂłth, Judit
Kiadó:
American Society for Biochemistry and Molecular Biology, Inc.
Dátum:
2016
Téma:
QD01 Analytical chemistry / analitikai kĂŠmia
Tartalmi leírás:
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.
Nyelv:
angol
Típus:
Article
PeerReviewed
info:eu-repo/semantics/article
Formátum:
text
Azonosító:
Lopata, Anna and Leveles, Ibolya and Bendes, Ábris Ádåm and Viskolcz, BÊla and G. VÊrtessy, Beåta and Jójårt, Balåzs and Tóth, Judit (2016) A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes. JOURNAL OF BIOLOGICAL CHEMISTRY, 291 (51). pp. 26320-26331. ISSN 0021-9258
Kapcsolat:
https://doi.org/10.1074/jbc.M116.734012
MTMT:3163582; doi:10.1074/jbc.M116.734012