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

项目摘要 这里提出的研究旨在解决PAS监管的结构和动态 蛋白激酶（人类中的hPASK），使用生物化学和生物物理方法的混合。时代-ARNT- PAS结构域，已知其感知广泛的环境线索以调节效应子 proteins. hPASK维持两个PAS结构域（PAS-A和PAS-B）的进化保守结构。 B）和典型丝氨酸/苏氨酸激酶（STK）结构域。 不能感知和响应细胞代谢状态和不适当的脂质积累是 对糖尿病、肥胖症和其他代谢疾病的发病机理至关重要。在酵母和哺乳动物中 在细胞中，hPASK在响应细胞代谢状态的营养感测和代谢信号传导中是重要的。 这些观察结果使hPASK成为代谢性疾病预防的极好治疗候选物。 然而，这些努力受到信号的不完整结构和机制表征的限制 在全长hPASK蛋白内的转导。赞助商凯文·H博士加德纳研究了 在细菌和人类系统中，PAS结构域等蛋白质，这导致了许多重要的 治疗发展的突破。这项研究计划包括在博士的监督下工作。 加德纳获得专业知识，将不同的结构生物学技术与生化数据相结合， 表征这种PAS调节的蛋白激酶的结构和功能方面。 具体的研究目标是：1）了解感觉PAS-A域的动力学， 表征其结构内的配体结合谱，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） 鉴定这些多结构域组装体在全长蛋白质结构内的结构取向。第一、 探索PAS-A中的蛋白质-配体相互作用（PLI），尖端高压（HP）NMR方法， 包括加德纳实验室开创的新技术和X射线晶体学，将被使用。二是利用 生物物理（NMR，FRET，MS）和功能生化分析，分子机制和PPI位点 将鉴定hPASK结构域之间的相互作用。最后，这些动力学及其结构基础之间的 PAS-A-配体复合物和PAS与激酶结构域之间的PPI将在 在配体存在和不存在的情况下使用冷冻-EM的全长hPASK。 研究PAS-A结构域内配体选择性的决定因素和hPASK的动力学 通过上述生物物理表征的PLIs和PPI将增强研究人员对如何实现 信号在发信号期间在全长hPASK内传播。此外，这些生物物理学见解将 揭示了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.