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逆转录酶的逆转录酶活性（HIV- RT）。 因为HIV-RT的逆转录酶活性尚无已知 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 抑制剂是对一种结构和催化机制的了解 酶。 尽管HIV-RT的三维结构已经 关于催化机制或位置的阐明知之甚少 在或 在催化过程中。 拟议的研究的目的是开始 解决这些问题。 我们建议在 HIV-RT的核苷酸和底漆/模板结合结构域并评估 它们使用定向诱变的重要性。 具体而言，我们建议在中使用一系列光活性DNA探针 底漆/模板配置，以标记氨基酸 HIV-RT的引物/模板结合域。 隔离和氨基酸 与这些探针交联的肽的测序将允许 鉴定参与引物/模板结合的氨基酸 或催化。 提议的标签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 在3'端包含二氧光活性核苷酸类似物。 一个 光活性3'二氧基核苷酸将允许氨基酸标记 在酶的3'末端附近，酶有催化 但无法合并DNTP。 交联与光活性 具有A形式结构（RNA模板）或B形式的底漆/模板 结构（DNA模板）将使我们能够解决是否是否 当HIV-RT准备使用时，使用不同的蛋白质构象 DNA和RNA依赖模式中的催化。 我们还建议使用光活性核苷酸类似物标记氨基 DNTP结合结构域中的酸。 肽的分离和测序 与核苷酸探针的交联将识别可能 参与底物结合和/或催化。 识别残留物 最有可能参与催化，光性标签 RT在催化能力上时将进行实验 带有3'二氧基终止底漆/模板的三元综合体和下一个 补充DNTP。 确定由 交联实验我们建议将定向的诱变使用 在交联的部位和附近更改氨基酸。 我们的 初步结果表明Tyr-318在DNTP结合域中。 因此，我们建议将Tyr-318更改为PHE，THR和GLY以评估 该氨基酸的重要性。 突变的RT将被描述为 确定每个突变对酶活性的影响，KD的KD 引物/模板和DNTP的结合以及AZT的Ki。 结果 这项研究将对广泛的长期目标很重要， 是有效和特定逆转录酶的合理设计 抑制剂。