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

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。NT激活Trk可导致细胞内酪氨酸残基磷酸化，并触发介导神经突生长、神经元分化或存活的下游信号通路。众所周知，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受体胞外部分的免疫球蛋白样结构域5 (TrkB-d5)，但也有证据表明，跨越Trk序列的结构域5和受体跨膜段之间的长柔性连接体区域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工作或与多个靶标成功相互作用。NT-4/5在与TrkB-d5结合时发生了从无序到有序的转变，并且两种蛋白质单独和在复合物中都表现出显著的构象交换，这些观察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。具体的目标。该提案概述了一项计划，以核磁共振（NMR）光谱作为我们的主要分析工具，对NT/Trk相互作用和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水平表征。我们的具体目标是：1)研究NT-4/5与TrkB结构域5的连接区c端之间的相互作用程度，并确定它们之间的具体联系；2)在ps-ns和ms时间尺度上测量和比较同位素标记的hNT-4/5在未结合状态下和与未标记的hTrkB-d5或hTrkB- d5L （TrkB结构域5与附加的连接区）的主链动力学。3)测量和比较同位素标记的hTrkB-d5或hTrkB-d5L在未结合状态和与未标记的hNT-4/5结合时的主链动力学。核磁共振实验将包括滴定以监测复合物形成时的化学位移变化，残余偶极偶联和氢交换实验以结构表征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、帕金森病、慢性疼痛和关节炎。人们对开发以神经营养因子为基础的治疗方法有广泛的兴趣，但成功取决于对神经营养因子的特异性和功能机制的全面掌握，以便产生副作用更少的选择性药物。这些研究的结果将首次提供NTs中蛋白质运动和结合选择性之间相关性的见解，并可能最终对进一步了解该信号过程的生物物理特性以及提高我们治疗神经系统疾病的能力至关重要。

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.