Abstract
Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416-Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining α-helix M', Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors. Based on our structural data, we analyzed the role of the p51 C terminus by evaluating selectively mutated p66/p51 heterodimers carrying (i) p51 truncations that encroach on α-M', (ii) alterations that interrupt the Asn-348-Tyr-427 interaction, and (iii) alanine substitutions throughout the region Phe-416-Pro-421. Collectively, our data support the notion that the p51 C terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.
Highlights
New crystallographic data for human immunodeficiency virus (HIV)-1 reverse transcriptase (RT) containing an RNA/DNA hybrid show important interactions involving the p51 C terminus
C-terminal p51 Truncations Stabilize the HIV-1 RT Heterodimer—In a previous study, we reported inhibition of ribonuclease H (RNase H) and tRNA-primed (Ϫ) strand strong-stop DNA synthesis activities of a mutant HIV-1 RT, whose p51 subunit contained a 13-residue C-terminal truncation removing Gln-428 – Phe-440 [25]
Our recently solved cocrystal structure of HIV-1 RT complexed with an RNA/DNA hybrid and in the presence of an NNRTI [23] indicated that Tyr-427 of p51 ␣-helix MЈ and Asn-348 of the -17/-18 connecting loop participate in hydrogen bonding, raising the possibility that this important interaction was destabilized when residues Gln428 –Phe-440 were removed from the p51 C terminus p51
Summary
New crystallographic data for HIV-1 RT containing an RNA/DNA hybrid show important interactions involving the p51 C terminus. Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. We have taken advantage of our recently solved structure of HIV-1 RT containing an NNRTI and a non-polypurine tract RNA/DNA hybrid (PDB ID 4B3O) that differs structurally from all previously reported RT-nucleic acid complexes and is compatible with RNA degradation [23] to evaluate the effect of point mutations, deletions, and alanine scanning mutagenesis on positioning of ␣-helix MЈ (Pro-421–Tyr-427), which constitutes the last structural element of the p51 subunit visible from crystallography. Our data provide a cautionary note for crystal engineering of HIV-1 RT when attempting to define contacts with RNA/DNA hybrids
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