Structure of unliganded HIV-1 reverse transcriptase at 2.7 å resolution: implications of conformational changes for polymerization and inhibition mechanisms

Hsiou, Y.; Ding, Jianping; Das, Kalyan; Clark, Arnold M.; Hughes, Stephen H.; Arnold, Edward

doi:10.1016/s0969-2126(96)00091-3

Cited by 281 publications

(365 citation statements)

References 27 publications

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“…analysed here, as well as by structures from other laboratories [where the p66 thumb has been observed to be folded down against the rest of the molecule (Rodgers et al, 1995;Hsiou et al, 1996)]. We conclude that (especially for the p66 thumb) crystal packing is the major factor determining the orientation of these domains, at least within the range we observe.…”

Section: Discussionsupporting

confidence: 75%

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

et al. 1998

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A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a highresolution limit of 3.7 ,~. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2,~. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

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Section: Discussionsupporting

confidence: 75%

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

et al. 1998

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Pata

Stirtan

Goldstein

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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We have determined the crystal structure of the HIV type 1 reverse transcriptase complexed with CP-94,707, a new nonnucleoside reverse transcriptase inhibitor (NNRTI), to 2.8-Å resolution. In addition to inhibiting the wild-type enzyme, this compound inhibits mutant enzymes that are resistant to inhibition by nevirapine, efavirenz, and delaviridine. In contrast to other NNRTI complexes where tyrosines 181 and 188 are pointing toward the enzyme active site, the binding pocket in this complex has the tyrosines pointing the opposite direction, as in the unliganded protein structure, to accommodate CP-94,707. This conformation of the pocket has not been observed previously in NNRTI complexes and substantially alters the shape and surface features that are available for interactions with the inhibitor. One ring of CP-94,707 makes extensive stacking interactions with tryptophan 229, one of the few residues in the NNRTI-binding pocket that cannot readily mutate to give rise to drug resistance. In this conformation of the pocket, mutations of tyrosines 181 and 188 are less likely to disrupt inhibitor binding. Modeling the asparagine mutation of lysine 103 shows that a hydrogen bond between it and tyrosine 188 could form as readily in the CP-94,707 complex as it does in the apoenzyme structure, providing an explanation for the activity of this inhibitor against this clinically important mutant.

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“…In nucleic acid-containing crystal structures, catalytic aspartates are close to the 3Ј terminus of the primer. Asp-185 and Asp-186 are part of the conserved -TyrMet-Asp-Asp-motif, which adopts an unusual ␤-turn conformation (29,35,48), possibly to promote their positioning for catalysis, whereas the Tyr-183 phenoxy side chain is involved in hydrogen bonding with nucleobases at position Ϫ2 (29). Comparison of the crystal structures of DNA-bound RT with unliganded or non-nucleoside RT inhibitor (NNRTI)-bound enzymes reveals significant conformational differences for the -Tyr-Met-Asp-Asp-motif, implicating a high degree of structural flexibility (29).…”

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confidence: 99%

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Grice

2012

Journal of Biological Chemistry

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Synthesis of integration-competent, double-stranded DNA from the (؉)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA-and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (؊)-and (؉)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here. HIV-1 DNA SynthesisThe individual steps of HIV-1 DNA synthesis, catalyzed by the multifunctional reverse transcriptase (RT), 2 are summarized schematically in Fig. 1. (Ϫ)-Strand DNA synthesis, initiated from a cellular tRNA (tRNA 3 Lys ) hybridized to the genomeencoded primer-binding site, continues to the 5Ј terminus, creating (Ϫ)-strong-stop DNA. RNase H-mediated degradation of the resulting RNA/DNA hybrid promotes relocation of nascent (Ϫ)-DNA to the genome 3Ј terminus by a strand transfer event that exploits sequence homology between the 5Ј and 3Ј termini. RNA-templated DNA synthesis continues, accompanied by RNase H-mediated degradation of the RNA genome, the exception to which are two short purine-rich segments (the 3Ј-and central polypurine tracts (PPTs)) from which (ϩ)-strand DNA-dependent DNA synthesis is initiated. Newly synthesized (Ϫ)-strand DNA and 18 nucleotides of the covalently attached tRNA 3 Lys primer provide the template for 3Ј-PPTprimed (ϩ)-strand DNA synthesis until the replication complex stalls at a position corresponding to the first modified tRNA base (A57). As a consequence, the C-terminal RNase H domain is positioned at the (Ϫ)-DNA/tRNA junction, and degradation of the tRNA "template" promotes a second or (ϩ)-strand transfer event supported by homology between (Ϫ)-and (ϩ)-strand DNA primer-binding sites. Although bidirectional DNA synthesis would be sufficient to complete DNA synthesis, HIV utilizes a second, central PPT primer, thereby producing a (ϩ)-strand discontinuity (1). Following (ϩ)-strand transfer, 3Ј-PPT-mediated DNA synthesis continues, displacing ϳ100 nucleotides of central PPT-primed (ϩ)-DNA, but abruptly ceases at the central termination sequence, a prominent feature of which is phased A-tracts that induce minor groove compression (1-3), creating the "central flap" (Fig. 1) (4 -7). Although central flap function remains controversial (8, 9), its mutation or deletion has been shown to impair...

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Structure of unliganded HIV-1 reverse transcriptase at 2.7 å resolution: implications of conformational changes for polymerization and inhibition mechanisms

Cited by 281 publications

References 27 publications

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

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

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

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

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