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

阐明 hPASK（一种 PAS 调节的哺乳动物激酶）的结构动力学

• 批准号: 10392342
• 负责人: Roksana Azad
• 金额: $ 3.25万
• 依托单位: ADVANCED SCIENCE RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392342
• 关键词: ARNT gene; Address; Allosteric Site; Architecture; Binding; Binding Sites; Biochemical; Biological Assay; Biological Process; Biophysics; Cells; Complex; Cryoelectron Microscopy; Cues; Data; Diabetes Mellitus; Disease; Exhibits; Failure; Fluorescence Resonance Energy Transfer; Goals; Human; Impairment; In Vitro; Length; Ligand Binding; Ligands; Light; Link; Lipids; Mammalian Cell; Metabolic; Metabolic Diseases; Methods; Molecular; Molecular Conformation; Mus; N-terminal; Obesity; Outcome; Pathogenesis; Phosphotransferases; Play; Point Mutation; Protein Binding Domain; Protein Dynamics; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Regulation; Research; Research Personnel; Research Project Summaries; Rest; Role; Sensory; Signal Pathway; Signal Transduction; Site; Structure; Supervision; System; Techniques; Testing; X-Ray Crystallography; Yeasts; biophysical properties; biophysical techniques; detection of nutrient; diabetes pathogenesis; disorder prevention; insight; insulin signaling; kinase inhibitor; mutant; pressure; protein protein interaction; protein structure; response; sensor; structural biology; therapeutic candidate; therapeutic development; therapeutic target

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.

项目概要 这里提出的研究旨在解决 PAS 监管的结构和动态 结合生物化学和生物物理方法的蛋白激酶（人类 hPASK）。 单心（PAS）域，已知可以感知广泛的环境线索来调节效应器 hPASK 维持两个 PAS 结构域（PAS-A 和 PAS-）的进化保守结构。 B) 和典型的丝氨酸/苏氨酸激酶 (STK) 结构域。 未能感知和响应细胞代谢状态和不适当的脂质积累是 对于酵母和哺乳动物的糖尿病、肥胖症和其他代谢疾病的发病机制至关重要。 在细胞中，hPASK 在响应细胞代谢状态的营养传感和代谢信号传导中发挥着重要作用。 这些观察结果使 hPASK 成为代谢性疾病预防的极佳候选治疗药物。 然而，这种努力受到信号结构和机械特性不完整的限制。 赞助者 Kevin H. Gardner 博士研究了全长 hPASK 蛋白内的转导。 细菌和人类系统中的 PAS 结构域等蛋白质，导致了许多重要的结果 该研究计划包括在博士的监督下进行工作。 加德纳将获得将不同结构生物学技术与生化数据相结合的专业知识 表征该 PAS 调节蛋白激酶的结构和功能。 具体研究目标是：1）了解感觉PAS-A域的动态，以及 表征其结构内的配体结合概况，2) 确定蛋白质- hPASK 结构域之间的蛋白质相互作用 (PPI) 位点的生物学功能和调节，以及 3) 首先，确定这些多域组装体在全长蛋白质结构中的结构方向。 探索 PAS-A 中的蛋白质-配体相互作用 (PLIs)、尖端高压 (HP) NMR 方法、 其次，将使用包括加德纳实验室首创的新技术和 X 射线晶体学。 生物物理（NMR、FRET、MS）和功能生化检测、分子机制和 PPI 检测位点 最后，将确定 hPASK 结构域之间的这些动态及其结构基础。 PAS-A-配体复合物以及 PAS 和激酶结构域之间的 PPI 将在 使用冷冻电镜在存在和不存在配体的情况下显示全长 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 上述通过生物物理表征得出的 PLI 和 PPI 将增强研究人员对如何 此外，信号在信号传导过程中在全长 hPASK 内传播。 揭示了 hPASK 作为代谢疾病治疗靶点的潜力。