The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1.

Abstract

The crystal structure of the reverse transcriptase (RT) from the type 1 human immunodeficiency virus has been determined at 3.2-A resolution. Comparison with complexes between RT and the polymerase inhibitor Nevirapine [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783Science 256, -1790 and between RT and an oligonucleotide [Jacobo-Molina, A., Ding, J., Nanni, R., Clark, A. D., Lu, X., Tantillo, C., Williams, R. L., Kamer a heterodimer (p66/p5i), with domains labeled "fingers," "thumb," "palm," and "connection" in both subunits, and a ribonuclease H domain in the larger subunit only. The most striking difference between RT and both complex structures is the change in orientation of the p66 thumb (-33°rotation). Smaller shifts relative to the core of the molecule were also found in other domains, including the p66 fingers and palm, which contain the polymerase active site. Within the polymerase catalytic region itself, there are no rearrangements between RT and the RT/DNA complex. In RT/Nevirapine, the drug binds in the p66 palm near the polymerase active site, a region that is well-packed hydrophobic core in the unliganded enzyme. Room for the drug is provided by movement of a small ,B-sheet within the palm domain of the Nevirapine complex. The rearrangement within the palm and thumb, as well as domain shifts relative to the enzyme core, may prevent correct placement of the oligonucleotide substrate when the drug is bound.The reverse transcriptase (RT) from the type 1 human immunodeficiency virus type 1 (HIV-1) is a heterodimer composed of a 66-kDa subunit (p66) and a 51-kDa subunit (pSi) derived from p66 by proteolytic removal of the C-terminal domain. RT possesses both DNA polymerase activity, which ultimately produces double-stranded DNA from the viral genomic RNA, and a ribonuclease H (RNase H) activity, which cleaves the viral genome after it is copied. A crystal structure of RT complexed with the drug Nevirapine (RT/ Nevirapine), a non-nucleoside-analog polymerase inhibitor, has been reported (1, 2), as well as the structure (3) of a complex with an 18/19-mer oligonucleotide (RT/DNA). We report here the structure of the unliganded enzyme at 3.2-A resolution." By comparing it with RT/Nevirapine and RT/ DNA, we can begin to examine the mechanisms of drug and nucleic acid binding. These processes are found to involve changes in domain arrangement within the enzyme but no major repositioning of the polymerase catalytic residues. Differences between the unliganded enzyme and RT/Nevirapine suggest a possible mechanism for the action of nonnucleoside inhibitors. MATERIALS AND METHODSCrystallization. Expression and purification of HIV-1 (BH10 strain) RT Data Collection. RT crystals used for data collection were dialyzed against buffer containing 50% ammonium sulfate, 60 mM sodium phosphate (pH 6.8), and 20% (vol/vol) glycerol. Crystals were mounted in loops (6) made from nylon fibers and flash-cooled in the gaseous nitrogen stream from a modified c...

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

“…From the results obtained, we conclude that the ␤ and Pol subunits fulfill mainly a structural function similar to that of the p51 subunits of HIV-1 RT and EIAV RT (13,14,19,21,29,32). However, we cannot completely exclude the possibility that the large subunit fulfills some minor function in catalysis, since mutations in Asp 181 or Asp 505 of the ␤ subunit appear to have some impact on the polymerase or RNase H activities.…”

“…amino acid residues of the polymerase and RNase H active sites of RTs are highly conserved (2,3,15). In addition, comparison of the crystal structures of HIV-1 RT and the finger and palm domains of murine leukemia virus RT indicates that the geometry of the polymerase active sites of RTs is highly conserved (6,14,19,29). We used this information for selecting the amino acids to be mutated in RSV RT to obtain enzymes impaired in their polymerase or RNase H activities.…”

Structure of HIV-1 reverse transcriptase bound to an inhibitor active against mutant reverse transcriptases resistant to other nonnucleoside inhibitors

“…4b) from the position that it occupies in DNA-bound HIV-1 RT complexes (51)(52)(53)(54)(55) and in unliganded HIV-1 RT structures crystallized in the absence of inhibitors (21,22,24). In other NNRTI complexes with HIV-1 RT, the displacement of Trp-229 is required for the rearrangements of Tyr-181 and Tyr-188 that occur upon inhibitor binding.…”

“…Subsequent structures of the enzyme with a number of different NNRTIs bound have demonstrated that these inhibitors bind to the same pocket, with only minor differences in protein conformation, and substantially overlap the site occupied by nevirapine (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Comparison of these structures with those of the apoenzyme shows that the NNRTI-binding pocket is induced upon NNRTI binding (21)(22)(23)(24).…”

Continuous and Discontinuous Changes in the Unit Cell of HIV-1 Reverse Transcriptase Crystals on Dehydration

“…Stammers et al, 1990;Unge et al, 1990;Lloyd et al, 1991;Kohlstaedt et al, 1992;Jones et al, 1993;Jacobo-Molina et al, 1993;Stammers et al, 1994;Rodgers et al, 1995), although few showed diffraction to high resolution. The structure was eventually described [for RT in complex with the NNI nevirapine (Merluzzi et al, 1990)] based on data to a minimum Bragg spacing of 3.4 A (Kohlstaedt et al, 1992), and was later extended to 2.9 A resolution .…”