期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 113, 期 33, 页码 9274-9279出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1605223113
关键词
EFdA; HIV-1 reverse transcriptase; inhibitors; NRTIs; X-ray crystallography
资金
- Office of Science, Office of Basic Energy Sciences, of the US Department of Energy (DOE) [DE-AC02-05CH11231]
- US DOE [DE-AC02-06CH11357]
- National Institutes of Health [AI076119, AI099284, AI100890, AI120860, GM103368, GM118012]
- Mizzou Advantage
- Trail to a Cure
- Grants-in-Aid for Scientific Research [16H05346] Funding Source: KAKEN
4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3'-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 angstrom) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNA(EFdA-MPP center dot dT-MPN)), or (iii) after incorporation of two EFdA-MPs (RT/DNA(EFdA-MPP center dot EFdA-MPN)); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNA(EFdA-MPP center dot EFdA-MP*N)). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4'-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.
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