NNRTI induced conformational changes in HIV-1 RT

描述(由申请人提供)： HIV-1逆转录酶(RT)是由66 kDa亚基(简称p66)和p66衍生的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)的活性完全取决于酶的四级结构。最近的研究表明，非核苷类RT抑制物(NNRTI)可以调节RT的p66和p51多肽之间的亚基间相互作用。在这一点上，NNRTI可以分为三个不同的组。第一组包括2‘，5’-双-O-(叔丁基二甲基硅烷)-beta-D-ribofuranosyl]-3‘spiro-5“-(4”-amino-1’‘2“-oxathiole-2”，2“-dioxide)胸腺嘧啶(TSAO-T)和N-酰肼的衍生物，破坏了HIV-1RT中亚单位间的相互作用。第二组包括奈韦拉平和法韦伦兹，增强HIV-1RT中亚单位间的相互作用。第三组，包括地拉韦定，没有任何效果。NNRTI与RT结合的分子机制可以调节酶的亚基间相互作用，以及这种调节对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-1RT亚基间相互作用和亚基内构象变化的机制。HIV-1RT p66/p51异源二聚体的形成是一个复杂的过程，涉及亚基之间的相互作用和亚基内的构象变化。基于荧光共振能量转移(FRET)或RT色氨酸荧光的分析系统将被开发来监测RT中的双分子蛋白质相互作用和单分子构象变化。然后，这些测试系统将被用于产生有关p66/p51 RT二聚化过程的定量热力学和动力学数据，以及NNRTI结合如何对其产生影响。(2)明确HIV-1逆转录二聚体界面的分子相互作用，并评价改变其固有的二聚体稳定性对酶活性的影响。与RT二聚体界面相比，NNRTI-BP很小，目前还不清楚NNRTI结合如何在整体上影响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也不确定。为了解决这些问题，这个目标寻求产生关于RT二聚体界面中靠近NNRTI-BP和从NNRTI-BP移除的氨基酸侧链的重要性的高分辨率功能数据。此外，包含稳定或破坏酶二聚体稳定性的突变的RT将被评估其执行DNA聚合酶和核糖核酸酶H(RNase H)活性的能力。我们的研究结果将为深入了解p66/p51 RT异源二聚体中蛋白质-蛋白质相互作用的性质以及NNRTI的作用机制提供重要的见解。

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.