Structural studies of PKR regulation by viral non-coding RNA

描述（由申请人提供）：PKR是细胞对抗病毒或细菌感染的先天免疫反应的关键组成部分，是控制其他不同细胞过程（如细胞周期、细胞生长、细胞凋亡和对细胞应激（如生长因子剥夺）的反应）的信号的重要中介和整合者。PKR功能异常也与人类疾病有关，包括癌症、神经退行性疾病以及潜在的代谢紊乱和肥胖。目前，由于缺乏高分辨率的PKR-RNA复合物结构信息，我们对RNA或其他分子调控PKR及其作为药物靶点的潜力的理解受到阻碍。本提案描述了一项研究计划，以获得双链RNA激活蛋白激酶（PKR）的晶体：非编码病毒RNA复合物，以产生这种重要的细胞激酶及其RNA调控的第一个高分辨率分子快照。我们应对这一重大挑战的方法分为两个综合目标。首先，由于RNA晶体学成功的最关键决定因素是RNA结构本身，因此Specific Aim 1将利用我们对非编码腺病毒转录物VA RNAI的详细生化理解，以产生用于PKR结晶的多种新型RNA构建物“文库”。这些包括RNA结构域的系统性变异，紧凑和稳定的RNA二级结构（如四环和四环受体）的引入，以及其他蛋白质（U1A或最近开发的RNA Fab）可以促进结晶的特定结合位点的结合。我们还开发了一种严格的策略，用于确保每种新型VA RNAI构建物保持野生型PKR抑制活性，包括通过紫外熔化进行全局RNA折叠分析，PKR自磷酸化抑制功能分析，以及分别通过凝胶过滤层析或等温滴定量热法对PKR-RNA结合进行定性或定量测量。Specific Aim 2将采用高通量自动化方法进行结晶和x射线衍射筛选，以产生必要的晶体。这一目标的初始实验将是迭代的，在结晶或低的第一次成功

DESCRIPTION (provided by applicant): PKR is a key component of the cellular innate immune response against viral or bacterial infection and is an important mediator and integrator of signals that control other diverse cellular processes such as the cell cycle, cell growth, apoptosis, and the response to cell stresses such as growth factor deprivation. Aberrant PKR function is also associated with human diseases including cancers, neurodegenerative diseases and, potentially, metabolic disorders and obesity. Our understanding of PKR regulation by RNA, or other molecules, and its potential as a drug target are currently hampered by the lack of high-resolution structural information for a PKR-RNA complex. This proposal describes a research program to obtain crystals of a double-stranded RNA-activated protein kinase (PKR):non-coding viral RNA complex in order to produce the first high-resolution molecular snapshot of this important cellular kinase and its regulation by RNA. Our approaches to this major challenge are grouped into two integrated aims. First, since the most critical determinant of success in RNA crystallography is the RNA construct itself, Specific Aim 1 will exploit the detailed biochemical understanding we have developed of the non-coding adenoviral transcript VA RNAI to produce a diverse 'library' of novel RNA constructs for crystallization with PKR. These include systematic variation of the RNA structural domains, introduction of compact and stable RNA secondary structures such as tetraloops and tetraloop receptors, and incorporation of specific binding sites for other proteins (U1A or a recently developed RNA Fab) that can promote crystallization. We have also developed a rigorous strategy that will be used to ensure that each of these novel VA RNAI constructs retains wild-type PKR-inhibition activity, including global RNA folding analysis by UV melting, PKR autophosphorylation inhibition functional assays, and qualitative or quantitative measurement of PKR-RNA binding by gel filtration chromatography or isothermal titration calorimetry, respectively. Specific Aim 2 will employ high-throughput automated approaches to crystallization and X-ray diffraction screening to produce the necessary crystals. Initial experiments in this aim will be iterative in that first successes in crystallization or low resolution structure determination will feed back into the approaches of Aim 1 to refine the RNA construct design and ultimately produce crystals suitable for high-resolution X-ray crystal structure determination. The structure of the PKR-RNA complex will be determined by molecular replacement using the PKR kinase domain structure, other protein (U1A or Fab), and/or RNA fragments as the starting model(s). Alternatively, selenomethionine incorporation into protein components of the complex will be used to obtain experimental phases to determine the structure. These studies will thus provide a critical structural breakthrough that is a prerequisit for mechanistic studies of PKR and the future development of this important enzyme as a therapeutic target via structure aided design of small molecule inhibitors.