Elucidating the structural dynamics of hPASK, a PAS regulated mammalian kinase

项目摘要 这里提出的研究试图解决受监管的PAS的结构和动态 使用生物化学和生物物理方法的混合方法来研究蛋白激酶(hPASK在人类中)。这一时期--阿尼特-- 单思维(PAS)结构域，已知可以感知广泛的环境线索来调节效应器 蛋白质。HPASK保持进化保守的两个PAS域(PAS-A和PAS-A)的体系结构 B)和一个典型的丝氨酸/苏氨酸激酶(STK)结构域。 未能感觉到细胞代谢状态和不适当的脂质积累并做出反应是 对糖尿病、肥胖症和其他代谢性疾病的发病机制至关重要。在酵母和哺乳动物中 HPASK在响应细胞代谢状态的营养感知和代谢信号中起重要作用。 这些观察结果使hPASK成为预防代谢性疾病的极佳候选治疗药物。 然而，这种努力受到信号的不完整的结构和机械表征的限制 全长hPASK蛋白内的转导。赞助商凯文·H·加德纳博士研究了 蛋白质，如细菌和人类系统中的PAS结构域，这导致了许多重要的 治疗开发取得突破性进展。这项研究计划包括在Dr。 Gardner将获得将不同的结构生物学技术与生化数据相结合的专业知识 描述这种PAS调节的蛋白激酶的结构和功能。 具体的研究目标是：1)了解感觉性PAS-A域的动力学，以及 表征其结构中的配基结合谱，2)确定蛋白质- HPASK结构域之间的蛋白质相互作用(PPI)位点，用于其生物学功能和调节，以及3)到 确定全长蛋白质结构中这些多结构域组件的结构方向。第一, 为了探索PAS-A中的蛋白质-配体相互作用(PLI)，前沿的高压(HP)核磁共振方法， 包括加德纳实验室开创的新技术和X射线结晶学，将被使用。第二，使用 生物物理(核磁共振、FRET、MS)和功能生化分析、分子机制和PPI位点 之间的hPASK域将被识别。最后，这些动态及其结构基础在 PAS-A-配基复合体以及PAS和激酶结构域之间的PPI将在 冷冻-EM法测定有无配体时的全长hPASK。 PAS-A结构域内配体选择性的决定因素及hPASK的动力学研究 通过上述生物物理特征描述的PLI和PPI将增强研究人员对如何 信号在信令期间在全长hPASK内传播。此外，这些生物物理洞察力将 阐明hPASK作为代谢性疾病治疗靶点的潜力。

Project Summary The research proposed here seeks to address the structure and dynamics of a PAS regulated protein Kinase (hPASK in humans) using a mix of biochemical and biophysical approaches. The Period-ARNT- Singleminded (PAS) domains, known to sense a wide range of environmental cues to regulate effector proteins. The hPASK maintains an evolutionarily conserved architecture of two PAS domains (PAS-A & PAS- B) and a canonical Serine/Threonine Kinase (STK) domain. The failure to sense and respond to cellular metabolic status and inappropriate lipid accumulation is critical to the pathogenesis of diabetes, obesity, and other metabolic diseases. In both yeast and mammalian cells, hPASK is important in nutrient sensing and metabolic signaling in response to cellular metabolic states. These observations make hPASK an excellent therapeutic candidate for metabolic disease prevention. However, such efforts are limited by incomplete structural and mechanistic characterization of signal transduction within the full length hPASK protein. The sponsor, Dr. Kevin H. Gardner has studied the sensory protein such as PAS domain in both bacterial and human systems, which has led to many significant breakthroughs in therapeutic development. This research plan includes working under the supervision of Dr. Gardner to gain expertise in integrating different structural biology techniques with biochemical data to characterize the structural and functional aspects of this PAS regulated protein kinase. The specific research goals are: 1) to understand the dynamics of the sensory PAS-A domain, and characterize the ligand binding profile within its structure, 2) to determine the Protein- Protein Interactions (PPIs) site between the hPASK domains for its biological function and regulation, and 3) to identify the structural orientation of these multidomain assemblies within the full length protein structure. First, to explore Protein-Ligand Interactions (PLIs) in PAS-A, cutting edge High Pressure (HP) NMR methods, including new techniques pioneered by the Gardner lab and X-ray crystallography, will be used. Second, using biophysical (NMR, FRET, MS) and functional biochemical assays, the molecular mechanism and PPIs sites between the hPASK domain will be identified. Finally, these dynamics and their structural basis between the PAS-A–ligand complex and the PPIs between the PAS and kinase domains will be further characterized within the full length hPASK in the presence and absence of ligand using cryo-EM. Studying the determinants of ligand selectivity within the PAS-A domain and the dynamics of hPASK PLIs and PPIs via biophysical characterization outlined above will enhance researchers understanding of how the signal propagates within the full length hPASK during signaling. Additionally, these biophysical insights will shed light on the potential of hPASK as a therapeutic target in metabolic diseases.