Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

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 A) 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-MP P • dT-MP N ), or ( iii ) after incorporation of two EFdA-MPs (RT/DNA EFdA-MP P • EFdA-MP N ); ( iv ) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNA EFdA-MP P • 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.