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.

项目摘要 这里提出的研究旨在解决受调节的PA的结构和动态 蛋白激酶（人类中的HPASK）使用生化和生物物理方法的混合物。周期 - 智慧（PAS）域，已知可以感知各种环境线索来调节效应器 蛋白质。 HPASK保持了两个PAS域的进化构成（PAS-A＆PAS-） b）和规范的丝氨酸/苏氨酸激酶（STK）结构域。 无法感知并应对细胞代谢状态和不适当的脂质积累的反应是 对于糖尿病，肥胖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 细胞，HPASK在响应细胞代谢态的养分感测和代谢信号传导中很重要。 这些观察结果使HPASK成为预防代谢疾病的出色治疗候选者。 但是，这种努力受信号的结构和机械表征不完整的限制 全长HPASK蛋白内的转导。赞助商凯文·H·加德纳（Kevin H. Gardner）博士已经研究了感官 细菌和人类系统中的蛋白质，例如PAS结构域，这导致了许多重要的 热发育中的突破。该研究计划包括在博士的监督下工作。 加德纳（Gardner 表征该PAS调控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领域的动态以及 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）位点的生物功能和调节，3） 确定这些多域组件在全长蛋白质结构内的结构取向。第一的， 为了探索PAS-A中的蛋白质 - 配体相互作用（PLIS），尖端高压（HP）NMR方法， 将使用由Gardner Lab和X射线晶体学开创的新技术。第二，使用 生物物理（NMR，FRET，MS）和功能性生化测定，分子机制和PPIS位点 将在HPASK域之间确定。最后，这些动态及其结构基础 PAS-A-配体配合物以及PAS和激酶结构域之间的PPI将进一步表征 使用冷冻EM的配体存在和不存在配体的全长HPASK。 研究PAS-A结构域内配体选择性的确定性和HPASK的动力学 通过以上概述的生物物理表征的PLI和PPI将增强研究人员了解如何 信号在信号传导过程中全长HPASK内传播。此外，这些生物物理见解将 阐明了HPASK作为代谢疾病的治疗靶标的潜力。