TY - JOUR
T1 - Structural basis of HIV inhibition by translocationdefective RT inhibitor 4′-ethynyl-2-fluoro-2′- deoxyadenosine (EFdA)
AU - Salie, Zhe Li
AU - Kirby, Karen A.
AU - Michailidis, Eleftherios
AU - Marchand, Bruno
AU - Singh, Kamalendra
AU - Rohan, Lisa C.
AU - Kodama, Eiichi N.
AU - Mitsuya, Hiroaki
AU - Parniak, Michael A.
AU - Sarafianos, Stefan G.
N1 - Funding Information:
We thank Jay Nix of Advanced Light Source beamline 4.2.2 for support. The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy (DOE) under Contract DE-AC02-05CH11231. We also acknowledge the Advanced Photon Source. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US DOE Office of Science by Argonne National Laboratory, was supported by the US DOE under Contract DE-AC02-06CH11357. This work was supported in part by grants from the National Institutes of Health (AI076119, AI099284, AI100890, AI120860, GM103368, and GM118012), Mizzou Advantage, and Trail to a Cure.
PY - 2016/8/16
Y1 - 2016/8/16
N2 - 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 Å) 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/DNAEFdA-MPP•dT-MPN), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MPP•dT-MPN); (iv) the latter was also solved with EFdAMP mismatched at the N site (RT/DNAEFdA-MPP•dT-MPN). 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.
AB - 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 Å) 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/DNAEFdA-MPP•dT-MPN), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MPP•dT-MPN); (iv) the latter was also solved with EFdAMP mismatched at the N site (RT/DNAEFdA-MPP•dT-MPN). 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.
KW - EFdA
KW - HIV-1 reverse transcriptase
KW - Inhibitors
KW - Nrtis
KW - X-ray crystallography
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U2 - 10.1073/pnas.1605223113
DO - 10.1073/pnas.1605223113
M3 - Article
C2 - 27489345
AN - SCOPUS:85047289150
VL - 113
SP - 9274
EP - 9279
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 33
ER -