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

HIV-1 RT RNase H 在溶液中的构象动力学和抑制剂反应

基本信息

批准号：
9302149
负责人：
RIEKO ISHIMA
金额：
$ 13.53万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2018-08-31
项目状态：
已结题

项目摘要

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.

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，但没有蛋白质核糖核酸酶H（RNH）活性的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，这可能导致难以以核糖核酸酶为治疗目的。直到最近才发表了与 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柔性区域，这对于底物识别和酶促反应非常重要。然而，这种灵活性如何影响抑制剂结合，反之亦然，尚未得到系统的研究。本研究的目标是在溶液中研究 RNHI 与 RNH 片段和全长 RT 的相互作用，在原子水平上。为此，将在 RNH 上进行溶液核磁共振波谱，结合生化和病毒学研究，使用 RNHI 作为探针来检测构象变化。拟议的研究将深入了解 RNHI 相互作用时的 RNH 结构域构象变化，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 是否是药物开发的可行目标。我们将完成以下具体目标：(1) 表征溶液中与活性位点 RNHI 相互作用时 RNH 构象变化，(2) 确定变构抑制的结构机制和 RNH 上的变构 RNHI 相互作用位点，(3) 检查相互作用(4) 使用定点诱变结合体外和体内测定验证结构发现。