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: 8274631
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8274631
• 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。神经营养因子（NT）蛋白通过与其同源Trk受体的相互作用来控制一些最基本的神经过程。通过NT激活Trk导致细胞内酪氨酸残基的磷酸化，并触发介导神经突生长、神经元分化或存活的下游信号传导途径。已知人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受体的细胞外部分中的免疫球蛋白样结构域5（TrkB-d5），但也有证据表明，跨越结构域5与受体的跨膜区段之间的Trk序列的长柔性接头区也可能在分子识别中起关键作用。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功能或与多个靶标成功相互作用。这些运动可能是结合选择性的关键组成部分的证据已经通过观察提供，即NT-4/5的部分在结合TrkB-d5时经历无序到有序的转变，并且两种蛋白质单独和复合物中显示出显著的构象交换。具体目标。该提案概述了一个计划，以追求一个彻底的，原子级的NT/Trk相互作用和蛋白质运动的结合选择性和分子识别的作用，使用核磁共振（NMR）光谱作为我们的主要分析工具。我们的具体目的是1）检查NT-4/5与TrkB上结构域5的C-末端的接头区之间的相互作用的程度，并鉴定它们之间的特异性接触，2）在ps-ns和ms时间尺度上测量并比较同位素标记的hNT-4/5在其未结合状态下的骨架动力学，以及当与未标记的hTrkB-d5或hTrkB-d5 L（具有连接的接头区的TrkB结构域5）结合时，以及3）测量并比较同位素标记的hTrkB-d5或hTrkB-d5 L在其未结合状态下以及当与未标记的hNT-4/5结合时的骨架动力学。NMR实验将包括滴定以监测复合物形成后的化学位移变化，残余偶极偶联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。健康相关的意义。NTs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的治疗方法来治疗这些疾病产生了广泛的兴趣，但成功取决于全面掌握神经营养因子的特异性和功能机制，以产生具有较少副作用的选择性药物。这些研究的结果将是第一个提供深入了解蛋白质运动和NT中结合选择性之间的相关性的结果，并且最终可能对于进一步理解这种信号传导过程的生物物理特性和提高我们治疗神经系统疾病的能力至关重要。 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参与信号传递的蛋白质内部运动与其识别并与参与信号传导途径的另一种蛋白质相互作用的能力之间的联系。如果我们能够对这一过程有更深入、更复杂的理解，我们可能会更好地使用设计成副作用更少的药物来治疗帕金森氏症、阿尔茨海默氏症或抑郁症等神经系统疾病。