Structure of HIV‐1 reverse transcriptase/d4TTP complex: Novel DNA cross‐linking site and pH‐dependent conformational changes

Abstract

Stavudine (d4T, 2′,3′‐didehydro‐2′,3′‐dideoxythymidine) was one of the first chain‐terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV‐1 RT/dsDNA template‐primer. We also present a new strategy for disulfide (S–S) chemical cross‐linking between N6 of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross‐link site is upstream of the duplex‐binding region of RT, however, the structure is very similar to published RT structures with cross‐linking to Q258C in the thumb, which suggests that cross‐linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X‐ray structures of the I63C‐RT/dsDNA/d4TTP cross‐linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH‐dependent motions of the fingers subdomain that folds in to form the dNTP‐binding pocket. We propose that the pH‐activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases.

Structural insights into the recognition of nucleoside reverse transcriptase inhibitors by HIV‐1 reverse transcriptase: First crystal structures with reverse transcriptase and the active triphosphate forms of lamivudine and emtricitabine

“…The 40 ) also leads to the conclusion that the peculiar binding mode of L-ddCTP, (−)FTC-TP, and (−) 3TC-TP is not caused from the missing second Mg +2 , from the missing 3 0 -hydroxyl of the compounds, nor from the difference in the ribose-like moiety. In fact, dCTP, (+)FTC-TP, D-ddCTP, and d4T-TP exhibit the same binding mode as the natural substrates, and TTP (PDB ID: 1RTD 35 ) despite the nonobservable second Mg +2 ion and the 3 0 -hydroxyl presence or lacking on the ribose-like heterocycle (Figures 4 and 5).…”

“…The comparison of the binding mode of L ‐ddCTP, (−)FTC‐TP, (+)FTC‐TP, and (−)3TC‐TP with the crystal structure of dCTP, D ‐ddCTP, and d4T‐TP in complex with RT (PDB ID: http://firstglance.jmol.org/fg.htm?mol=6AMO ) also leads to the conclusion that the peculiar binding mode of L ‐ddCTP, (−)FTC‐TP, and (−)3TC‐TP is not caused from the missing second Mg +2 , from the missing 3′‐hydroxyl of the compounds, nor from the difference in the ribose‐like moiety. In fact, dCTP, (+)FTC‐TP, D ‐ddCTP, and d4T‐TP exhibit the same binding mode as the natural substrates, and TTP (PDB ID: http://firstglance.jmol.org/fg.htm?mol=1RTD ) despite the nonobservable second Mg +2 ion and the 3′‐hydroxyl presence or lacking on the ribose‐like heterocycle (Figures and ).…”

Post-Catalytic Complexes with Emtricitabine or Stavudine and HIV-1 Reverse Transcriptase Reveal New Mechanistic Insights for Nucleotide Incorporation and Drug Resistance

“…The structure of the ternary complex of RT together with chain terminated dsDNA and d4T-TP inhibitor was previously elucidated by Martinez et al [ 14 ]. However, it was reported that changes in the pH value during crystallization resulted in motion of the finger region [ 14 ].…”

“…The structure of the ternary complex of RT together with chain terminated dsDNA and d4T-TP inhibitor was previously elucidated by Martinez et al [ 14 ]. However, it was reported that changes in the pH value during crystallization resulted in motion of the finger region [ 14 ]. Therefore, to be able to compare the binding modes of the different inhibitor under similar conformations of the protein, we reproduced this crystal structure at pH 6.0, the same pH as the other structure in this study.…”

“…Additionally, structural studies of HIV-1 RT by X-ray crystallography have elucidated the mechanism of reverse transcription initiation and incorporation of NRTIs. The combination of these studies has provided insight to how drug resistance to individual NRTIs develops [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Each NRTI acts as competitive inhibitor for the natural dNTP at the same binding site in RT; however, distinct mechanisms for discrimination and drug resistance have been observed.…”