NNRTI induced conformational changes in HIV-1 RT

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）活性完全依赖于酶的四级结构。最近的研究表明，非核苷类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。第一类，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”-氧硫杂环戊烯-2”，2”-二氧化物）胸腺嘧啶（TSAO-T）和N-酰基腙使HIV-1 RT中的亚单位间相互作用不稳定。第二组，包括奈韦拉平和依法韦仑，增强HIV-1 RT中亚单位间的相互作用。第三组，包括地拉韦啶，没有效果。NNRTI与RT结合可调节酶的亚基间相互作用的分子机制，以及这种调节对RT酶功能的影响尚不清楚。为此，本提案中所述的项目包括两个具体目标。(1)确定NNRTI调节HIV-1 RT亚基间相互作用和亚基内构象变化的机制。HIV-1 RT p66/p51异二聚体的形成是一个复杂的过程，涉及亚基间的相互作用和亚基内的构象变化。将开发基于荧光共振能量转移（FRET）或RT色氨酸荧光的检测系统，以监测RT中的双分子蛋白质-蛋白质相互作用和单分子构象变化。然后，这些检测系统将用于生成关于p66/p51 RT二聚化过程的定量热力学和动力学数据，以及NNRTI结合对其的影响。定义HIV-1 RT二聚体界面中的分子相互作用，并评价改变固有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结合如何全面影响RT中的亚基间相互作用。也不确定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，这一目标旨在产生高分辨率的功能数据的重要性，在RT二聚体界面附近的氨基酸侧链，并删除，NNRTI-BP。此外，将评估含有使酶的二聚体稳定性稳定或不稳定的突变的RT进行DNA聚合酶和核糖核酸酶H（RNA酶H）活性的能力。我们的研究结果应该提供显着的洞察的性质，蛋白质-蛋白质相互作用的p66/p51 RT异源二聚体和NNRTI的行动机制。

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.