Defining the architecture of the Pyk2 activation complex

神经元通过突触连接与邻近的神经元交流。这种神经元的“线路”可以随着时间的推移而改变和适应。控制神经元连接变化的机制的许多方面仍然是谜。然而，加强和削弱神经元连接的能力是至关重要的，因为这种重新布线与学习和记忆的形成有关。每个突触连接都承载着大量的蛋白质，这些蛋白质准备好解释神经元通信的模式。该研究项目旨在发现一组蛋白质如何对突触活动作出反应，从而发出突触变化的信号。信号小组的中心枢纽是Pyk2，一种蛋白激酶。Pyk2通过重组其结构来激活，从而与相关的信号因子形成一个大的信号组装体。本研究的目的是确定Pyk2信号组装的结构。了解Pyk2信号团队的组装和激活将有助于深入了解神经元重新布线是如何在分子尺度上启动的。该提案将通过以团队为基础的概念培养研究生和本科生，研究生和本科生将建立顾问/被顾问关系。该项目的目的是揭示Pyk2信号连接突触后Ca2+内流和调节突触可塑性的Src级联的分子机制。阐明支架信号复合物的结构仍然是理解突触可塑性的前沿目标。然而，多蛋白信号复合物通常是大而高动态的，具有挑战性的结构表征目标。虽然已经确定了信号效应器的孤立片段的结构，但仍然存在一个关键问题：这些片段如何组装成功能性的信号复合物？该项目采用几种策略来剖析Pyk2非受体酪氨酸激酶的激活信号复合物。通过利用氢/氘交换质谱法，蛋白质相互作用和构象变化将被绘制。单粒子电子显微镜将阐明活化复合物的整体形状。这些方法适用于描述可访问的构象和体系结构的异质集成。由互补的生物物理方法得出的结构限制将被整合，以建立负责激活Pyk2的信号复合体的高阶结构模型。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理集群的支持。

Neurons communicate with neighboring neurons through synaptic connections. This neuronal 'wiring' can change and adapt over time. Many aspects of the mechanisms controlling the changes in neuronal connections remain enigmatic. Nevertheless, the ability to strengthen and weaken neuron connections is critically important because this rewiring is linked to learning and memory formation. Each synaptic connection hosts a large collection of proteins poised to interpret patterns of neuronal communication. This research project seeks to discover how one team of proteins responds to synaptic activity to signal for synaptic changes. The central hub of the signaling team is Pyk2, a protein kinase. Pyk2 activates by rearranging its structure to form a large signaling assembly with associated signaling factors. The goal of this research is to determine the architecture of the Pyk2 signaling assembly. Understanding the assembly and activation of the Pyk2 signaling team will yield insights into how neuronal rewiring is initiated at the molecular scale. This proposal will train graduate and undergraduate students by using a team-based concept where graduate and undergraduate students will have an advisor/advisee relationship.The objectives of this project are to reveal the molecular mechanisms of Pyk2 signaling linking post-synaptic Ca2+ influx to the Src cascades that tune synaptic plasticity. Illuminating the architecture of scaffolded signaling complexes remains a frontier goal in understanding synaptic plasticity. However, multi-protein signaling complexes are typically large and highly dynamic, challenging targets for structural characterization. While structures have been determined for isolated pieces of the signaling effectors, a critical question remains: how do the pieces assemble into functional signaling complexes? This project employs several strategies to dissect the activational signaling complex of the Pyk2 non-receptor tyrosine kinase. By leveraging hydrogen/deuterium exchange mass spectrometry, protein interactions and conformational changes will be mapped. Single particle electron microscopy will illuminate the overall shape of the activation complex. These approaches are amenable to describing heterogeneous ensembles of accessible conformers and architectures. Structural restraints derived by the complementary biophysical approaches will be integrated to build a model of the higher-order architecture of the signaling complex responsible for activating Pyk2. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.