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。神经营养素 (NT) 蛋白通过与其同源 Trk 受体相互作用来控制一些最基本的神经过程。 NT 激活 Trk 会导致细胞内酪氨酸残基磷酸化，并触发下游信号通路，介导神经突生长、神经元分化或存活。已知人类 NT-4/5 与其同源 Trk 受体之间相互作用的主要位点发生在 Trk 受体 (TrkB-d5) 胞外部分的免疫球蛋白样结构域 5，但也有证据表明跨越结构域 5 和受体跨膜片段之间 Trk 序列的长柔性接头区域也可能在分子识别中发挥关键作用。参与信号转导的蛋白质通常需要结构灵活性才能正常发挥作用或与多个靶标成功相互作用。通过观察 NT-4/5 的部分在结合 TrkB-d5 时经历无序到有序的转变以及两种蛋白质单独和复合物都显示出显着的构象交换，提供了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。该提案概述了一项计划，即使用核磁共振 (NMR) 光谱作为我们的主要分析工具，对 NT/Trk 相互作用以及蛋白质运动在结合选择性和分子识别中的作用进行彻底的原子级表征。我们的具体目标是 1) 检查 NT-4/5 和 TrkB 上结构域 5 的连接区域 C 端之间相互作用的程度，并确定它们之间的特定接触，2) 测量和比较 ps 上的主链动态-ns 和 ms 时间尺度，同位素标记的 hNT-4/5 在其未结合状态下，以及当与未标记的 hTrkB-d5 或 hTrkB-d5L 结合时（TrkB 结构域 5 附有连接子区域），3）测量并比较同位素标记的 hTrkB-d5 或 hTrkB-d5L 在未结合状态下以及与未标记的 hNT-4/5 结合时的主链动力学。 NMR 实验将包括滴定以监测复合物形成时的化学位移变化、残余偶极耦合和氢交换实验以在结构上表征 TrkB/NT 相互作用，以及自旋弛豫实验以分析 NT-4/5 和 TrkB 在多个时间尺度上的蛋白质运动。与健康相关的意义。 NT 的突变和修饰与许多疾病有关，包括阿尔茨海默病和帕金森病、慢性疼痛和关节炎。人们对开发基于 NT 的疗法来治疗这些疾病有着广泛的兴趣，但成功取决于对神经营养素的特异性和功能机制的全面掌握，以便产生副作用较少的选择性药物。这些研究的结果将首次深入了解 NT 中蛋白质运动和结合选择性之间的相关性，并且最终可能对于进一步了解该信号传导过程的生物物理特性并提高我们治疗神经系统疾病的能力至关重要。 公共健康相关性：我们的最终目标是在最基本的分子水平上了解参与信号传输的蛋白质内部运动与其识别参与信号传导途径的另一种蛋白质并与之相互作用的能力之间的联系。如果我们能够促进对这一过程的改进和更复杂的理解，我们也许能够使用副作用较少的药物更好地治疗帕金森氏症、阿尔茨海默氏症或抑郁症等神经系统疾病。