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）：通常将HIV逆转录酶（RT）聚合酶活性的抑制剂用于治疗目的，但没有蛋白质的核糖核酸酶H（RNH）活性的抑制剂可用于临床应用。有关RNH抑制剂（RNHI）如何与原子水平的RT相互作用的信息很少，这可能有助于将核糖核酸酶靶向治疗目的。直到最近，RNH活性位点抑制剂的晶体结构与RT或发表的RNH片段中的复合物中，尚未进行非活性位点RNHI的结构分析。理解RNH抑制的原子机制的一部分困难与RNH的构象柔韧性有关：RNH结构域具有相对较大的柔性区域，对于底物识别和酶促反应很重要。但是，这种柔韧性如何影响抑制剂结合，反之亦然，尚未以系统的方式研究。拟议的研究的目的是研究RNHI与RNH片段的相互作用，并在原子水平上以全长RT的形式进行解决方案。为此，将使用RNHIS作为检测构象变化的探针进行溶液NMR光谱，并与生化和病毒学研究一起进行RNH进行。拟议的研究将提供对RNHI相互作用时RNH结构域构象变化的洞察力，对RNHIS与溶液中与RNH相互作用的知识以及对RNHI结合口袋在构象上的构象如何稳定的理解。此类信息将有助于确定RNH是否是药物开发的可行靶标。 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体内测定。