Structural studies of PKR regulation by viral non-coding RNA

描述（由申请人提供）：PKR是针对病毒或细菌感染的细胞先天免疫应答的关键组分，并且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）的应答）的信号的重要介体和整合体。异常PKR功能还与人类疾病相关，包括癌症、神经退行性疾病以及潜在的代谢紊乱和肥胖。我们对PKR通过RNA或其他分子的调节及其作为药物靶点的潜力的理解目前受到PKR-RNA复合物缺乏高分辨率结构信息的阻碍。该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结晶学成功的最关键决定因素是RNA构建体本身，因此Specific Aim 1将利用我们对非编码腺病毒转录物VA RNAi的详细生物化学理解来产生用于与PKR一起结晶的新型RNA构建体的多样化“文库”。这些包括RNA结构域的系统性变化，引入紧凑和稳定的RNA二级结构，如四环和四环受体，以及掺入其他蛋白质（U1 A或最近开发的RNA Fab）的特异性结合位点，可以促进结晶。我们还开发了一种严格的策略，将用于确保这些新的VA RNAi构建体中的每一个保留野生型PKR抑制活性，包括通过UV熔解进行的全局RNA折叠分析、PKR自磷酸化抑制功能测定以及分别通过凝胶过滤色谱法或等温滴定量热法进行的PKR-RNA结合的定性或定量测量。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，以生产必要的晶体。在这一目标的初步实验将是迭代的，在第一次成功的结晶或低 高分辨率结构测定将反馈到目标1的方法中，以改进RNA构建体设计，并最终产生适合于高分辨率X射线晶体结构测定的晶体。PKR-RNA复合物的结构将通过使用PKR激酶结构域结构、其他蛋白质（U1 A或Fab）和/或RNA片段作为起始模型的分子置换来确定。或者，硒代甲硫氨酸掺入到复合物的蛋白质组分中将用于获得实验阶段以确定结构。因此，这些研究将提供一个关键的结构突破，这是PKR的机制研究的先决条件，并通过小分子抑制剂的结构辅助设计，这种重要的酶作为治疗靶点的未来发展。 公共卫生相关性：该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，该复合物将产生这种重要细胞激酶及其RNA调控的第一个高分辨率分子结构快照。这种结构是PKR未来详细结构-功能研究及其作为主要治疗靶点发展的先决条件。

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. PUBLIC HEALTH RELEVANCE: This proposal describes a research program to obtain crystals of a double-stranded RNA-activated protein kinase (PKR):non-coding viral RNA complex that will produce the first high-resolution molecular structural snapshot of this important cellular kinase and its regulation by RNA. This structure is a prerequisite for future detailed structure-function studies of PKR and its development as a major therapeutic target.