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）聚合酶活性抑制剂通常用于治疗目的，但没有蛋白质核糖核酸酶H（RNH）活性抑制剂可用于临床应用。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，这可能导致难以靶向核糖核酸酶用于治疗目的。直到最近才公布了与RT或RNH片段复合的RNH活性位点抑制剂的晶体结构，并且尚未进行非活性位点RNHI的结构分析。理解RNH抑制的原子机制的部分困难与RNH的构象灵活性有关：已知RNH结构域具有相对较大的灵活区域，这些区域对于底物识别和酶促反应很重要。然而，这种灵活性如何影响抑制剂结合，反之亦然，还没有被系统地研究。拟议的研究的目标是调查RNHI与RNH片段和全长RT的相互作用，在原子水平上，在溶液中。为此目的，溶液NMR光谱，与生物化学和病毒学研究，将进行RNH，使用RNHI作为探针，以检测构象变化。拟议的研究将提供深入了解RNHI相互作用后的RNH结构域构象变化，RNHI在溶液中与RNH相互作用的知识，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是否是药物开发的可行靶点。我们将完成以下具体目标：（1）表征在溶液中与活性位点RNHI相互作用后RNH构象的变化，（2）鉴定RNH上的变构抑制的结构机制和变构RNHI相互作用位点，（3）检查RNHI与溶液中全长RT的相互作用，和（4）使用结合体外和体内测定的位点特异性诱变来验证结构发现。

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.