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; 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）是由66 kDa亚基（称为p66）和p66派生的51 kDa亚基（p51）组成的异二聚体酶。 RT的DNA聚合酶和核糖核酸酶H（RNase H）活性完全取决于酶的第四纪结构。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可以分为3个不同的组。 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。该提案中描述的项目包括两个特定的目的，以确定NNRTI调节HIV-1 RT互动的相互作用和subunit构象变化。将开发或RT色氨酸荧光，以监测RT中的双子分子蛋白 - 蛋白质相互作用和单分子构象变化。然后，这些测定系统将用于生成有关p66/p51 RT二聚过程的定量热力学和动力学数据，以及NNRTI结合如何影响它。 （2）定义HIV-1 RT二聚体界面中的分子相互作用，并评估改变固有二聚体稳定性对酶活性的后果。与RT二聚体界面相比，NNRTI-BP很小，尚不理解NNRTI结合如何在全球范围内影响RT中的亚基间相互作用。 NNRTI介导的RT抑制是否是调节酶的亚基间相互作用的直接结果。为了解决这些问题，此目标旨在生成有关RT二聚体界面中氨基酸侧链在附近并从NNRTI-BP中删除的高分辨率功能数据。此外，将评估包含稳定或破坏酶稳定性的突变的RT，以评估其执行DNA聚合酶和核糖核酸酶H（RNase H）活性的能力。我们的研究结果应提供对p66/p51 RT异二聚体中蛋白质蛋白相互作用的性质的重大见解和NNRTI作用的机制。