NNRTI induced conformational changes in HIV-1 RT

NNRTI 诱导 HIV-1 RT 构象变化

• 批准号: 6785443
• 负责人: NICOLAS PAUL SLUIS-CREMER
• 金额: $ 23.97万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6785443
• 关键词: DNA directed DNA polymerase; RNA directed DNA polymerase; conformation; dimer; enzyme activity; enzyme structure; fluorescence resonance energy transfer; genetic screening; human immunodeficiency virus 1; protein protein interaction; reverse transcriptase inhibitors; ribonuclease H; thermodynamics; tryptophan

DESCRIPTION (provided by applicant): HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme consisting of a 66-kDa subunit (termed p66) and a p66- derived 51-kDa subunit (p51). The DNA polymerase and ribonuclease H (RNase H) activities of RT are entirely dependent on the quaternary structure of the enzyme. Recent studies have shown that nonnucleoside RT inhibitors (NNRTI) can modulate the inter-subunit interactions between the p66 and p51 polypeptides of RT. In this regard, NNRTI can be classified into 3 distinct groups. The first group, which includes derivatives of 2',5'-Bis-O-(tert-butyldimethylsilyi)- beta-D-ribofuranosyl]-3'spiro-5"-(4"-amino-1''2"-oxathiole-2",2"-dioxide) thymine (TSAO-T) and N-acyl hydrazones, destabilize the inter-subunit interactions in HIV-1 RT. The second group, which includes nevirapine and efavirenz, enhance the inter-subunit interactions in HIV-1 RT. The third group, which includes delavirdine, elicits no effect. The molecular mechanisms by which NNRTI binding to RT can modulate the inter-subunit interactions of the enzyme, and the impact that this modulation has on RT enzymatic functioning is not known. To this end, the project described in this proposal comprises two Specific Aims. (1) To determine the mechanism by which NNRTI modulate HIV-1 RT intersubunit interactions and intra-subunit conformational changes. HIV-1 RT p66/p51 heterodimer formation is a complex process that involves inter-subunit interactions and intra-subunit conformational changes. Assay systems based on fluorescence resonance energy transfer (FRET) or RT tryptophan fluorescence will be developed that monitor the bimolecular protein-protein interactions and unimolecular conformational changes in RT. These assay systems will then be used to generate quantitative thermodynamic and kinetic data regarding the p66/p51 RT dimerization process and how NNRTI-binding can impact on it. (2) To define the molecular interactions in the HIV-1 RT dimer interface and to evaluate the consequences of altering the intrinsic dimeric stability on enzymatic activity. The NNRTI-BP is small compared to the RT dimer interface and it is not understood how NNRTI binding can globally affect the inter-subunit interactions in RT. It is also not certain as to whether the NNRTI-mediated inhibition of RT is a direct result of modulating the enzyme's inter-subunit interactions. To address these issues, this aim seeks to generate high-resolution functional data regarding the importance of amino acid side chains in the RT dimer interface near to, and removed from, the NNRTI-BP. Furthermore, RT containing mutations that either stabilize or destabilize the dimeric stability of the enzyme will be assessed for their capacity to carry out both DNA polymerase and ribonuclease H (RNase H) activities. The results of our studies should provide significant insight into the nature of the protein-protein interactions in the p66/p51 RT heterodimer and the mechanisms of NNRTI action.

描述（由申请人提供）： HIV-1 逆转录酶 (RT) 是一种异二聚体酶，由 66 kDa 亚基（称为 p66）和 p66 衍生的 51 kDa 亚基（p51）组成。 RT 的 DNA 聚合酶和核糖核酸酶 H (RNase H) 活性完全取决于酶的四级结构。最近的研究表明，非核苷 RT 抑制剂 (NNRTI) 可以调节 RT 的 p66 和 p51 多肽之间的亚基间相互作用。在这方面，NNRTI 可分为 3 个不同的组。第一组，包括2',5'-双-O-(叔丁基二甲基甲硅烷基)-β-D-呋喃核糖基]-3'螺-5"-(4"-氨基-1''2"-的衍生物氧硫醇-2",2"-二氧化物）胸腺嘧啶 (TSAO-T) 和 N-酰基腙会破坏 HIV-1 RT 中亚基间相互作用的稳定性。奈韦拉平和依非韦伦增强了 HIV-1 RT 中亚基间的相互作用。这种调节对 RT 酶功能的影响尚不清楚。为此，本提案中描述的项目包括两个具体目标 (1) 确定 NNRTI 调节 HIV-1 RT 的机制。 HIV-1 RT p66/p51 异二聚体形成是一个复杂的过程，涉及基于荧光共振能量转移 (FRET) 或 RT 色氨酸荧光的亚基间相互作用和亚基内构象变化。将开发监测 RT 中双分子蛋白质-蛋白质相互作用和单分子构象变化的技术。然后，这些检测系统将用于生成有关 p66/p51 RT 二聚化过程以及 NNRTI 结合如何影响它的定量热力学和动力学数据。 (2) 定义 HIV-1 RT 二聚体界面中的分子相互作用，并评估改变内在二聚体稳定性对酶活性的影响。与 RT 二聚体界面相比，NNRTI-BP 较小，目前尚不清楚 NNRTI 结合如何全局影响 RT 中亚基间的相互作用。还不确定 NNRTI 介导的 RT 抑制是否是调节酶亚基间相互作用的直接结果。为了解决这些问题，该目标旨在生成关于靠近 NNRTI-BP 和从 NNRTI-BP 移除的 RT 二聚体界面中氨基酸侧链重要性的高分辨率功能数据。此外，将评估含有稳定或破坏酶二聚体稳定性的突变的 RT 执行 DNA 聚合酶和核糖核酸酶 H (RNase H) 活性的能力。我们的研究结果将为 p66/p51 RT 异二聚体中蛋白质-蛋白质相互作用的性质以及 NNRTI 作用机制提供重要的见解。