Structures of HIV-1 RT-RNA/DNA ternary complexes with dATP and nevirapine reveal conformational flexibility of RNA/DNA: insights into requirements for RNase H cleavage

Abstract

In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2′-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal that conformational changes of an RNA/DNA and increased interactions with the RNase H domain, including the interaction of a 2′-OH with N474, help to position the RNA nearer to the active site. The structures and existing biochemical data suggest a nucleic acid conformation-induced mechanism for guiding cleavage of the RNA strand.

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

“…The cross‐linked RT139A (I63C) cocrystallized with bound d4TTP in the P2 1 space group and with cell dimensions similar to those of previously reported Q258C RT/nucleic acid cross‐linked complexes . While Q258C RT/DNA crystals (binary or ternary complex) grow at approximately pH 6.8–7.6, I63C RT/DNA/d4TTP crystals only grow over the pH range 9.5–10.5; attempts to grow at a lower pH generated only spherules.…”

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

“…Due to the lack of a 3′‐OH on the chain‐terminated DNA primer and the NRTIs, the Mg +2 ion A binding is weakened and may not have full occupancy in the crystal structures to be observed. This is not surprising as Mg +2 ion A is detected in only a few crystal structures of RT in complex with dsDNA and in complex with DNA/RNA hybrid . The three phosphate groups are firmly anchored in the active site by an extensive hydrogen bond network involving additionally R72, D110, D113, A114, D185, and the Mg +2 ion.…”

Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications

“…Notably, the positioning of the α1′ helix of the GsI-IIC RT NTE sterically precludes the RNA-DNA duplex in our structure from adopting the same conformation as RNA-DNA duplexes in HIV-1 and TERT (Das et al, 2014; Mitchell et al, 2010). The insertion of this helix into the major groove widens the groove and increases the distance between the n+2 and p+9 phosphates to 13.5 Å compared to 11.2 Å for HIV-1 RT (Fig.…”