Structural studies of PKR regulation by viral non-coding RNA

描述(申请人提供)：PKR是针对病毒或细菌感染的细胞先天免疫反应的关键组成部分，也是控制其他不同细胞过程的信号的重要介体和整合者，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。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PKR-RNA复合体的高分辨率结构信息，我们对RNA或其他分子对PKR的调控及其作为药物靶点的潜力的理解受到阻碍。这项建议描述了一项研究计划，以获得双链RNA激活的蛋白激酶(PKR)的晶体：非编码的病毒RNA复合体，以产生第一个高分辨率的分子快照这一重要的细胞激酶及其由RNA调节。我们应对这一重大挑战的方法归结为两个综合目标。首先，由于RNA结晶学中最关键的决定因素是RNA结构本身，因此特定目标1将利用我们对非编码腺病毒转录物VA RNAi的详细生化理解来产生一个多样化的新RNA结构的“库”，用于与PKR一起结晶。这些措施包括RNA结构域的系统性变异，引入紧凑稳定的RNA二级结构，如Tetraloop和Tetraloop受体，以及加入其他蛋白质(U1a或最近开发的RNA Fab)的特定结合部位，以促进结晶。我们还制定了一套严格的策略，以确保每个新的VA RNAi结构物都保持野生型PKR抑制活性，分别包括通过UV熔融进行全局RNA折叠分析，通过PKR自动磷酸化抑制功能分析，以及通过凝胶过滤层析或等温滴定量热法定性或定量测定PKR-RNA结合。特殊目标2将使用高通量自动化方法进行结晶和X射线衍射筛选，以生产必要的晶体。这一目标初步实验将在结晶或更低的第一次成功中反复进行 分辨率结构测定将反馈到目标1的方法，以改进RNA结构设计，并最终生产适合于高分辨率X射线晶体结构测定的晶体。PKR-RNA复合体的结构将以PKR激酶结构域结构、其他蛋白质(U1a或Fab)和/或RNA片段为起始模型通过分子置换来确定(S)。或者，将硒蛋氨酸掺入到复合体的蛋白质组分中，以获得确定结构的实验相。因此，这些研究将为PKR的机制研究和通过小分子抑制剂的结构辅助设计将这一重要酶作为治疗靶点的未来发展提供关键的结构突破。

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.