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

简介(申请人提供)：背景。神经营养因子(NT)蛋白通过与其同源Trk受体相互作用来控制一些最基本的神经过程。NT激活Trk导致细胞内酪氨酸残基的磷酸化，并触发下游信号通路，介导轴突生长、神经元分化或存活。已知，人NT-4/5与其同源Trk受体相互作用的主要部位发生在Trk受体胞外部分(TrkB-d5)的免疫球蛋白样区5，但也有证据表明，位于Trk区5和受体跨膜片段之间的长柔性连接区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上的灵活性才能正常发挥作用或成功地与多个靶点相互作用。有证据表明，这些运动可能是结合选择性的关键成分，这是因为观察到NT-4/5的部分在与TrkB-d5结合时经历了无序到有序的转变，并且这两种蛋白质单独和在复合体中都显示出显著的构象交换。明确的目标。该提案概述了一项计划，利用核磁共振光谱作为我们的主要分析工具，对这种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水平表征。我们的具体目标是1)检查NT-4/5与TrkB上的连接区C-末端到结构域5之间的相互作用的程度并识别它们之间的特定接触，2)测量和比较同位素标记的hNT-4/5在其未结合状态下以及当与未标记的hTrkB-d5或hTrkB-d5L(TrkB结构域5与连接的连接区)结合时的骨架动力学，以及3)测量和比较同位素标记的hTrkB-d5或hTrkB-d5L在其未结合状态下的骨架动力学，核磁共振实验将包括监测络合物形成时的化学位移变化的滴定实验，从结构上表征TrkB/NT相互作用的剩余偶极耦合和氢交换实验，以及分析NT-4/5和TrkB中多个时间尺度的蛋白质运动的自旋弛豫实验。与健康相关的意义。NTS的突变和修改与许多疾病有关，包括阿尔茨海默氏症和帕金森氏症、慢性疼痛和关节炎。开发基于NT的疗法来治疗这些疾病被广泛关注，但成功取决于全面掌握神经营养素的特异性和功能机制，以便生产出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NTS中蛋白质运动和结合选择性之间的关系，并最终可能对我们进一步了解这一信号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。 与公共健康相关：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参与信号通路的另一种蛋白质并与其相互作用的能力之间的联系。如果我们能够对这一过程做出更好、更复杂的理解，我们可能就能更好地使用设计为具有较少负面副作用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。

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.