ACTIVE SITE OF HIV-1 REVERSE TRANSCRIPTASE

HIV-1 逆转录酶的活性位点

• 批准号: 2185741
• 负责人: ROBERT K EVANS
• 金额: $ 8.24万
• 依托单位: WILLIAMS COLLEGE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185741
• 关键词: Baculoviridae; DNA; Insecta; RNA; RNA directed DNA polymerase; active sites; affinity labeling; chemical binding; chemical kinetics; crosslink; enzyme activity; gene mutation; human immunodeficiency virus 1; nucleic acid probes; nucleotide analog; protein sequence; recombinant proteins; site directed mutagenesis; tissue /cell culture; zidovudine

Replication of the Human Immunodeficiency virus is totally dependent on the reverse transcriptase activity of HIV-1 reverse transcriptase (HIV- RT). Because the reverse transcriptase activity of HIV-RT has no known cellular homolog it is an important target for the rational design of antiviral drugs. The basis of the rational design of specific enzyme inhibitors is a knowledge of the structure and catalytic mechanism of an enzyme. Although the three dimensional structure of HIV-RT has been elucidated little is known about the catalytic mechanism or the locations of residues that play critical roles in substrate binding before or during catalysis. The goal of the proposed research is to begin addressing these issues. We propose to identify amino acids in the nucleotide and primer/template binding domains of HIV-RT and to evaluate their importance using site-directed mutagenesis. Specifically, we propose to use a series of photoactive DNA probes in primer/template configurations, to label amino acids in the primer/template binding domain of HIV-RT. Isolation and amino acid sequencing of peptides crosslinked to these probes will allow the identification of amino acids that participate in primer/template binding or in catalysis. Two key features of the proposed labeling experiments include the use of both RNA and DNA templates, and the use of DNA primers containing a dideoxy photoactive nucleotide analog at the 3' end. A photoactive 3' dideoxy nucleotide will allow the labeling of amino acids near the 3' end of the primer while the enzyme is poised for catalysis but unable to incorporate dNTPs. Crosslinking to photoactive primer/templates having an A-form structure (RNA template) or a B-form structure (DNA template) will allow us to address the question of whether different protein conformations are used when HIV-RT is poised for catalysis in the DNA and RNA dependent modes. We also propose to use photoactive nucleotide analogs to label amino acids in the dNTP binding domain. Isolation and sequencing of peptides crosslinked to the nucleotide probes will identify amino acids likely to be involved in substrate binding and/or catalysis. To identify residues most likely to be involved in catalysis, photoaffinity labeling experiments will be performed while RT is in a catalytically competent ternary complex with a 3' dideoxy terminated primer/template and the next complementary dNTP. To determine the importance of the amino acids identified by the crosslinking experiments we propose to use site-directed mutagenesis to change amino acids at and near the sites of crosslinking. Our preliminary results suggest that Tyr-318 is in the dNTP binding domain. Therefore, we propose to change Tyr-318 to Phe, Thr and Gly to evaluate the importance of this amino acid. Mutant RTs will be characterized to determine the effects of each mutation on enzyme activity, the Kd for binding of primer/template and dNTPs and the Ki for AZT. The results of this study will be important toward the broad long-term objective, which is the rational design of potent and specific reverse transcriptase inhibitors.

人类免疫缺陷病毒的复制完全取决于 HIV-1逆转录酶（HIV-1）的逆转录酶活性 转发）。 因为 HIV-RT 的逆转录酶活性尚不清楚 细胞同源物是合理设计的重要目标 抗病毒药物。 特定酶合理设计的基础 抑制剂是关于抑制剂的结构和催化机制的知识。 酶。 尽管 HIV-RT 的三维结构已被 对催化机制或位置知之甚少 在底物结合之前或之前起关键作用的残基 催化过程中。 拟议研究的目标是开始 解决这些问题。 我们建议鉴定氨基酸 HIV-RT 的核苷酸和引物/模板结合域并进行评估 使用定点诱变来认识它们的重要性。 具体来说，我们建议使用一系列光活性 DNA 探针 引物/模板配置，以标记氨基酸 HIV-RT 的引物/模板结合域。 分离和氨基酸 对与这些探针交联的肽进行测序将允许 鉴定参与引物/模板结合的氨基酸 或在催化作用中。 所提出的标记实验的两个关键特征 包括使用 RNA 和 DNA 模板，以及使用 DNA 引物 3'端含有双脱氧光活性核苷酸类似物。 一个 光活性 3' 双脱氧核苷酸将允许标记氨基酸 靠近引物 3' 端，同时酶准备催化 但无法合并 dNTP。 交联至光活性 具有A型结构（RNA模板）或B型结构的引物/模板 结构（DNA 模板）将使我们能够解决以下问题： 当 HIV-RT 准备进行时，会使用不同的蛋白质构象 DNA 和 RNA 依赖模式下的催化。 我们还建议使用光活性核苷酸类似物来标记氨基 dNTP 结合域中的酸。 肽的分离和测序 与核苷酸探针交联将识别可能的氨基酸 参与底物结合和/或催化。 识别残留物 最有可能参与催化、光亲和标记 实验将在 RT 处于催化能力的情况下进行 具有 3' 双脱氧终止引物/模板的三元复合物和下一个 互补 dNTP。 确定氨基酸鉴定的重要性 交联实验我们建议使用定点诱变 改变交联位点及其附近的氨基酸。 我们的 初步结果表明 Tyr-318 位于 dNTP 结合域中。 因此，我们建议将Tyr-318改为Phe、Thr和Gly来评估 这种氨基酸的重要性。 突变 RT 的特征是 确定每个突变对酶活性的影响，即 Kd 引物/模板和 dNTP 的结合以及 AZT 的 Ki。 结果 这项研究对于实现广泛的长期目标非常重要， 是有效且特异的逆转录酶的合理设计 抑制剂。