Use of a combinatorial disulfide crosslinking strategy to prepare a stalled complex of HIV-1 reverse transcriptase (RT) with a DNA template/primer and a nucleoside triphosphate (dNTP) has led to a crystal structure at 3.2 Å resolution. Presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the molecular basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chemical structure of the drug.

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Download the crystal structure of the  HIV Reverse Transcriptase / DNA template : primer / dNTP complex

Positional variations explored in the combinatorial search for efficient disulfide cross-linking between RT and DNA. The relative positions of the mutated cysteine and the tethered thiol group in DNA were varied (i) by introducing cysteine into successive turns of helix H (blue) and (ii) by having the polymerization reaction terminate after a defined number of cycles of the reaction (brown), thereby ratcheting RT forward by a defined distance (cyan) on the DNA substrate. This approach created 12 combinations of relative positions of the reacting thiol groups on RT and DNA.

The reverse transcriptase (RT) of HIV-1 is an important target of antiviral therapy in the treatment of acquired immunodeficiency syndrome (AIDS). RT has two distinct enzymatic activities, an RNA- or DNA-dependent DNA polymerase and a ribonuclease (RNase) H, but current agents are directed only against the polymerase. Five of the seven inhibitors currently licensed in the United States are chain-terminating nucleoside analogs [for example, 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and 2'-deoxy-3'-thiacytidine (3TC)]. The other two inhibitors are members of a chemically diverse group of nonnucleoside RT inhibitors (NNRTIs).

HIV-1 RT is a dimer of two related chains, a 66-kD subunit (p66) and a 51-kD subunit (p51) derived from p66 by proteolytic cleavage. The two chains have in common four domains [referred to as "fingers," "palm," "thumb," and "connection"], and p66 also has a COOH-terminal RNase H. The p66 subunit has both the polymerase and RNase H active sites. The palm contains residues critical for polymerase catalytic activity, and its folded structure resembles that of a corresponding catalytic domain in other DNA and RNA polymerases.

SDS-PAGE analysis of the reactions between the three engineered cysteine mutants and the thiol-bearing template:primer. In the nonreducing PAGE used here with protein staining, disulfide cross-linking between the p66 subunit of RT and DNA results in the appearance of a new band having retarded mobility (p66-DNA), accompanied by reduced intensity of the p66 band itself.

Emergence of resistance in patients treated with RT inhibitors is a major limitation of antiviral therapy. All NNRTIs bind near the polymerase active site, in a hydrophobic pocket created by displacement of the polypeptide segment connecting palm and thumb. Viral mutations conferring resistance to these drugs can be understood readily in terms of alterations in their common binding site. In contrast, the positions of altered residues in viruses resistant to nucleoside analogs do not follow so clear a pattern. Lack of a structure for a catalytic complex of RT with template:primer and dNTP substrates has hindered understanding how site-specific changes confer resistance to particular drugs.

Previously determined RT structures include a number of NNRTI complexes and the unliganded enzyme. The only published structure of RT with a bound template:primer is an RT-template:primer-Fab ternary complex, which shows that the primer terminus lies near three catalytically essential aspartic acid residues in the palm and that the duplex extends along the enzyme surface toward the RNase H. The position of fingers and thumb define a deep cleft, with the polymerase active site at its base. This feature, also present in crystals of RT with bound NNRTIs, has led to a model in which the 5' extension of the template passes through the cleft, interacting with residues in the fingers and palm that are mutated in drug-resistant strains. Other DNA polymerases contain a similar cleft, but recent structures of catalytic complexes show that it closes down when substrates bind and that it does not serve as a channel for polynucleotide chains.

The active site. (To the left) View into the dNTP pocket, formed by closure of the fingers domain against the palm and toward the thumb.  Template and primer strands and dTTP are in stick representation; the protein is a surface rendering (red and blue indicate negative and positive electrostatic potential, respectively); Mg ions are yellow spheres.

We have devised a way of isolating stalled, covalently tethered complexes of RT with template:primer, and we have crystallized one such species. In the complex described here at 3.2 ?nbsp;resolution, the primer terminus is a dideoxynucleotide and thus unable to attack an incoming dNTP. The crystal of this catalytic complex contains bound dTTP in precisely the expected position for attack by the (missing) 3' OH. The fingers domain bends, relative to other RT structures, so that various residues near the fingertips form part of the dNTP-binding site. This conformational adjustment defines the complete catalytic site and leads to a revised interpretation of the mechanism by which various mutations confer resistance to nucleoside analog drugs.

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