STRUCTURES OF HIV REVERSE TRANSCRIPTASE WITH SUBSTRATES

已开发了一套灵活的系统来研究结构 HIV-1逆转录酶与核酸模板的复合体 引发剂和抑制剂的分辨率为2.8埃。到目前为止，我们已经 确定了HIV-1RT与A的三元复合体的结构 单抗Fab片段(FAB28)和19/18碱基dsDNA (19/18 dsDNA)3.5埃分辨率的模板-引物，正在完善中 该结构的分辨率为2.8埃。我们所处的水晶形态 使用特别有利，因为1)它提供了第一个 以某种方式与核酸结合的任何聚合酶的原子结构 与聚合有关，以及2)含有抗病毒的复合体 可以研究化合物、不同的模板-引物和HIV-1 RT突变体 通过直接的差分傅里叶方法。因为只有更改过的 结构的一部分需要解决，所有拟议的研究 可以用相应的工具来完成。 具体地说，使用HIV-1RT/FAB28晶体，我们将解决和提炼 含有三种不同核酸的变异体的结构 模板-引物(19/18dsDNA、30/18dsDNA和30/18RNA/DNA)。这些 RT/Fab/模板-引物复合体将成为所有 其他决定的结果。我们将确定 含三磷酸型核苷的RT/FAB/DNA晶体 使用野生型和核苷类抗药性艾滋病毒的抑制剂AZT和DDI- 1个RT突变体。HIV-1/HIV-2嵌合体的结构 多聚酶活性位点已被移植到HIV-1 RT将被 确定，从而允许检查HIV-2 RT的结构 活动站点。 从这项工作中获得的结构信息将有助于 了解DNA聚合的分子机制 对核苷抑制剂的抑制和抗药性的发展。这个 核苷三磷酸抑制剂的详细立体化学描述 HIV-1RT的复合体在设计和提纯HIV-1RT中应该有价值 抗病毒药物。

A flexible system has been developed for studying the structures of complexes of HIV-1 reverse transcriptase (RT) with nucleic acid template- primers and inhibitors at 2.8 Angstrom resolution. We have thus far determined the structure of a ternary complex of HIV-1 RT with a monoclonal antibody Fab fragment (Fab28) and a 19 base/18 base dsDNA (19/18 dsDNA) template-primer at 3.5 Angstrom resolution and are refining the structure at 2.8 Angstrom resolution. The crystal form that we are using is particularly advantageous because 1) it is providing the first atomic structure of any polymerase bound to nucleic acid in a mode relevant for polymerization, and 2) complexes containing antiviral compounds, different template-primers, and HIV-1 RT mutants can be studied by the straightforward difference Fourier method. Since only the altered portion of the structure needs to be solved, all of the proposed studies can be done with relative facility. Specifically, using the HIV-1 RT/Fab28 crystals, we will solve and refine the structures of variants that contain three different nucleic acid template-primers (19/18 dsDNA, 30/18 dsDNA, and 30/18 RNA/DNA). These RT/Fab/template-primer complexes will be the reference structures for all of the other determinations. We will determine the structures of the RT/Fab/DNA crystals containing triphosphate forms of the nucleoside inhibitors AZT and ddI, using both wild-type and nucleoside-resistant HIV- 1 RT mutants. The structure of an HIV-1 /HIV-2 chimera in which the HIV-2 polymerase active site has been transplanted onto HIV-1 RT will be determined, thus permitting examination of the structure of the HIV-2 RT active site. The structural information gained from this work will be helpful in understanding the molecular mechanisms of DNA polymerization, RT inhibition, and development of resistance to nucleoside inhibitors. The detailed stereochemical description of nucleoside triphosphate inhibitor complexes with HIV-1 RT should be valuable in the design and refinement of antiviral agents.