Conformational Dynamics and inhibitor responses of HIV-1 RT RNase H in solution

描述(由申请人提供)：HIV逆转录酶(RT)聚合酶活性的抑制剂通常用于治疗目的，但没有可用于临床应用的蛋白质核糖核酸酶H(RNH)活性的抑制剂。关于RNH抑制剂(RNHI)如何在原子水平上与RT相互作用的信息很少，这可能导致靶向核糖核酸酶用于治疗目的的困难。直到最近，RNH活性中心抑制剂与RT或RNH片段形成的络合物的晶体结构才被发表，而非活性中心RNHI的结构分析还没有进行。理解RNH抑制的原子机制的困难部分与RNH的构象灵活性有关：RNH结构域具有相对较大的柔性区域，对于底物识别和酶反应是重要的。然而，这种灵活性如何影响抑制剂结合，反之亦然，还没有系统地研究。拟议研究的目标是在原子水平上研究RNHI与RNH片段和全长RT在溶液中的相互作用。为此，将结合生化和病毒学研究对RNH进行溶液核磁共振光谱分析，使用RNHI作为探针来检测构象变化。拟议的研究将提供对RNHI相互作用时RNH结构域的构象变化的洞察，了解RNHI在溶液中与RNH相互作用的位置，以及了解RNHI结合口袋如何变得构象稳定。这些信息将有助于确定RNH是否为药物开发的可行目标。我们将完成以下特定目标：(1)表征RNH在溶液中与活性部位RNHI相互作用时的构象变化，(2)确定变构抑制的结构机制和RNH上的变构RNHI相互作用部位，(3)检测RNHI与溶液中全长RT的相互作用，以及(4)通过结合体外和体内试验的定点突变来验证结构发现。

DESCRIPTION (provided by applicant): Inhibitors of HIV reverse transcriptase (RT) polymerase activity are routinely used for therapeutic purposes, yet no inhibitor of the protein's ribonuclease H (RNH) activity is available for clinical application. Information on how RNH inhibitors (RNHI) interact with RT, at an atomic level, is scarce, and this may contribute to the difficulty in targeting the ribonuclease for therapeutic purposes. Only recently were crystal structures of RNH active-site inhibitors in complex with RT or an RNH fragment published, and structural analysis of non active- site RNHI has not been carried out. Part of the difficulty in understanding the atomic mechanisms of RNH inhibition relates to the conformational flexibility of RNH: the RNH domain is known to have relatively large flexible regions that are important for substrate recognition and the enzymatic reaction. Yet, how this flexibility influences inhibitor binding, and vice versa, has not been studied in a systematic manner. The goal of the proposed research is to investigate RNHI interactions with an RNH fragment and with full-length RT, at an atomic level, in solution. For this purpose, solution NMR spectroscopy, in concert with biochemical and virological studies, will be carried out on RNH, using RNHIs as probes to detect conformational changes. The proposed research will provide insight into RNH domain conformational changes upon RNHI interaction, knowledge of where RNHIs interact with RNH in solution, and an understanding of how RNHI binding pockets become conformationally stable. Such information will be useful for determining whether RNH is a feasible target for drug development. We will complete the following specific aims: (1) Characterize RNH conformational changes upon interaction with active-site RNHIs in solution, (2) Identify the structural mechanism of allosteric inhibition and the allosteric RNHI interaction site on RNH, (3) Examine the interactions of RNHIs with full-length RT in solution, and (4) Validate the structural findings using site-specific mutagenesis coupled with in vitro and in vivo assays.