Investigating protein dynamics in NT-4/5 and TrkB receptor interactions

研究 NT-4/5 和 TrkB 受体相互作用的蛋白质动力学

• 批准号: 8098224
• 负责人: KARIN A CROWHURST
• 金额: $ 14.36万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8098224
• 关键词: Academic Research Enhancement Awards; Acceleration; Address; Adverse effects; Affinity; Alzheimer' s Disease; Area; Arthritis; Binding; Brain-Derived Neurotrophic Factor; C-terminal; Chemicals; Complement Factor B; Complex; Coupling; Data Analyses; Development; Disease; Drug usage; Educational process of instructing; Funding; Future; Goals; Grant; Health; Human; Hydrogen; Immunoglobulin Domain; Investigation; Label; Light; Link; Measures; Mediating; Mental Depression; Mentors; Modification; Molecular; Molecular Conformation; Monitor; Motion; Mutation; NMR Spectroscopy; Neurites; Neurologic; Neuronal Differentiation; Neurotrophic Tyrosine Kinase Receptor Type 2; Neurotrophin 3; Nuclear Magnetic Resonance; Paper; Parkinson Disease; Pharmaceutical Preparations; Phosphorylation; Pilot Projects; Play; Process; Property; Protein Dynamics; Proteins; Publications; Recruitment Activity; Regulation; Relaxation; Research; Research Project Grants; Residual state; Role; Sampling; Scientist; Series; Shipping; Ships; Side; Signal Pathway; Signal Transduction; Site; Specificity; Structure; System; Testing; Therapeutic; Time; Titrations; Transmembrane Domain; Tyrosine; United States National Institutes of Health; Vertebral column; analytical tool; base; chronic pain; computerized data processing; data exchange; design; electron density; extracellular; flexibility; grasp; improved; insight; interest; member; molecular dynamics; molecular recognition; nervous system disorder; neuronal growth; neuronal survival; neurotrophic factor; neurotrophin 4; physical property; protein function; public health relevance; receptor; receptor binding; research study; success; transmission process

DESCRIPTION (provided by applicant): Background. Neurotrophin (NT) proteins control some of the most fundamental neurological processes through interactions with their cognate Trk receptors. Activation of a Trk by an NT results in phosphorylation of the intracellular tyrosine residues and triggers downstream signaling pathways that mediate neurite outgrowth, neuronal differentiation or survival. It is known that the primary site of interaction between human NT-4/5 and its cognate Trk receptor takes place at the immunoglobulin-like domain 5 in the extracellular portion of the Trk receptor (TrkB-d5), but there is also evidence that the long flexible linker region spanning the Trk sequence between domain 5 and the transmembrane segment of the receptor may also play a critical role in molecular recognition. Proteins involved in signal transduction often require structural flexibility to function properly or interact successfully with multiple targets. Evidence that these motions could be critical components to binding selectivity has been provided by observations that portions of NT-4/5 undergo disorder-to-order transitions upon binding TrkB-d5 and that both proteins display significant conformational exchange alone and in complex. Specific aims. The proposal outlines a plan to pursue a thorough, atomic-level characterization of this NT/Trk interaction and the role of protein motions in binding selectivity and molecular recognition using Nuclear Magnetic Resonance (NMR) spectroscopy as our primary analytical tool. Our specific aims are to 1) examine the extent of the interactions between NT-4/5 and the linker region C-terminal to domain 5 on TrkB and identify specific contacts between them, 2) measure and compare the backbone dynamics, on the ps-ns and ms timescales, of isotopically labeled hNT-4/5 in its unbound state, and when bound to unlabeled hTrkB-d5 or hTrkB- d5L (TrkB domain 5 with attached linker region), and 3) measure and compare the backbone dynamics of isotopically labeled hTrkB-d5 or hTrkB-d5L in its unbound state, and when bound to unlabeled hNT-4/5. NMR experiments will include titrations to monitor chemical shift changes upon complex formation, residual dipolar coupling and hydrogen exchange experiments to structurally characterize the TrkB/NT interactions, and spin relaxation experiments to analyze protein motions in NT-4/5 and TrkB at multiple timescales. Health-related significance. Mutations and modifications to NTs have been linked to numerous illnesses, including Alzheimer's and Parkinson's disease, chronic pain and arthritis. There is widespread interest in developing NT-based therapeutics to treat these conditions, but success is dependent on having a comprehensive grasp of the mechanism of specificity and function inneurotrophins in order to yield selective drugs with fewer side effects. The results of these studies will be the first to provide insight into the correlation between protein motions and binding selectivity in NTs and may ultimately be critical for furthering our understanding of the bio- physical properties of this signaling process and improving our ability treat neurological diseases. PUBLIC HEALTH RELEVANCE: Our ultimate goal is to understand, at the most fundamental molecular level, the connection between internal motions within a protein involved in signal transmission and its ability to recognize and interact with another protein that is participating in the signaling pathway. If we can contribute to an improved and more sophisticated understanding of this process, we may be better able to treat neurological diseases such as Parkinson's, Alzheimer's or depression using drugs that have been designed to have fewer negative side effects.

描述（由申请人提供）：背景。神经营养蛋白（NT）蛋白通过与同源TRK受体的相互作用来控制一些最基本的神经系统过程。通过NT激活TRK会导致细胞内酪氨酸残基和触发介导神经突产物，神经元分化或存活的下游信号传导途径的磷酸化。 It is known that the primary site of interaction between human NT-4/5 and its cognate Trk receptor takes place at the immunoglobulin-like domain 5 in the extracellular portion of the Trk receptor (TrkB-d5), but there is also evidence that the long flexible linker region spanning the Trk sequence between domain 5 and the transmembrane segment of the receptor may also play a critical role in molecular recognition.参与信号转导的蛋白质通常需要结构灵活性才能正常运行或成功与多个目标相互作用。观察到，NT-4/5的一部分在结合TRKB-D5时进行疾病到阶的转变，并且两种蛋白质单独并在复杂中显示出显着的构象交换，证明这些运动可能是结合选择性的关键组成部分的证据。具体目标。该提案概述了该NT/TRK相互作用的彻底，原子级表征的计划，以及使用核磁共振（NMR）光谱作为我们的主要分析工具，蛋白质运动在结合选择性和分子识别中的作用。我们的具体目的是1）检查NT-4/5与TRKB上的连接器区域C末端5之间的相互作用程度（TRKB结构域5具有连接的接头区域）和3）测量和比较其无绑定状态的同位素标记的HTRKB-D5或HTRKB-D5L的骨干动力学，以及当绑定到未标记的HNT-4/5时。 NMR实验将包括滴定，以监测复杂形成时的化学位移变化，残留的偶极耦合和氢交换实验，以在结构上表征TRKB/NT相互作用，并进行自旋松弛实验，以分析多个时间尺度的NT-4/5和TRKB中的蛋白质运动。与健康有关的意义。对NTS的突变和修饰与许多疾病有关，包括阿尔茨海默氏病和帕金森氏病，慢性疼痛和关节炎。人们普遍兴趣开发基于NT的治疗剂来治疗这些疾病，但成功取决于对特异性和功能不神经营养的机制的全面掌握，以产生副作用较少的选择性药物。这些研究的结果将是第一个提供有关NTS蛋白质运动和结合选择性之间相关性的见解，最终对于我们对我们对这种信号过程的生物物理特性的理解至关重要，并改善了我们的能力治疗神经疾病。 公共卫生相关性：我们的最终目标是在最基本的分子水平上了解信号传输涉及的蛋白质内部运动之间的联系及其与参与信号通路的另一种蛋白质识别和相互作用的能力。如果我们能够对这一过程有了改进，更复杂的理解，我们可能会更好地治疗诸如帕金森氏症，阿尔茨海默氏症或抑郁症之类的神经系统疾病，使用旨在减少负面副作用的药物。