Structure-function investigations of protein partnerships governing neuronal signaling

化学和电信号通过一种称为突触的特殊连接在神经元之间传递。为了使突触的每一侧有效地发挥作用，数百种受体、细胞内信息和细胞骨架蛋白需要以高度调节但动态的方式聚集在一起。这种流体状态的通信是通过支架和衔接蛋白实现的。我使用高分辨率结构生物学方法来确定支架和适配器如何使用蛋白质-蛋白质相互作用和翻译后修饰来帮助神经元在分子水平上建立连接并对外部刺激做出反应。 在这个建议中，以前发表的结构，从我的小组对衔接蛋白HACS 1和支架蛋白CASKIN 2扩展到非常规蛋白质的伙伴关系，由他们介导的调查。 HACS 1是一种模块化蛋白，其结构有助于它与信号转导和细胞骨架蛋白形成伙伴关系。目的1建立在先前确定的高分辨率结构的HACS 1 Src同源性3（SH 3）域从我的小组和发现，它与配对受体（PIRB）胞质区域以一种新的方式相互作用。这种相互作用被认为是新颖的，因为SH 3结构域结合表面的氨基酸互补序列以及识别的PIRB中的序列是非典型的。这项研究的长期目标是为来自PIRB的负信号如何通过HACS 1抑制神经元的成熟提供结构性解释。除了其SH 3结构域之外，HACS 1还具有无菌α基序（SAM）结构域，该结构域也以其支持广泛的蛋白质-蛋白质相互作用的能力而闻名。目标2的重点是为HACS 1 SAM领域发现新的合作伙伴。确定这些合作伙伴是建立完整的HACCP功能说明的重要的第一步1。在我的职业生涯中，我已经确定了许多SAM结构域的结构，我可以与我的研究小组合作，推动最有趣的合作伙伴进行高分辨率的结构研究。 与HACS 1相比，CASKIN 2的组织更复杂，因此被称为分子支架，而不是接头。CASKIN 2和CASKIN 1的相似之处在于它们在囊泡运输和在运动神经元和肌肉细胞之间建立适当连接中的作用。在目标3中，我将确定CASKIN 2的SAM结构域如何与LAR受体磷酸酶的已知但定义不清的区域相互作用的结构基础。选择CASKIN 2/LAR伙伴关系是因为SAM结构域被预测以不同于我已经解决的其他SAM结构域结构的方式与LAR相互作用。从这个目标的生化和结构的见解可以扩展到Liprin类的神经元支架，也使用SAM域结合LAR。

Chemical and electrical signals are transmitted between neurons through a specialized junction called a synapse. For each side of the synapse to function efficiently, hundreds of receptors, intracellular messages and cytoskeletal proteins need to be brought together in highly regulated, yet dynamic way. This fluid state of communication is achieved by scaffolding and adaptor proteins. I use high resolution structural biology methods to determine how scaffolds and adaptors use protein-protein interactions and post-translational modifications to help neurons make connections and respond to external stimuli at the molecular level. In this proposal, previously published structures from by my group on the adaptor protein HACS1 and the scaffolding protein CASKIN2 are extended to investigations of unconventional protein partnerships mediated by them. HACS1 is a modular protein whose architecture helps it form partnerships with signal transduction and cytoskeletal proteins. Aim 1 builds upon a previously determined high resolution structure of the HACS1 Src homology-3 (SH3) domain from my group and the discovery that it interacts with the Paired receptor (PIRB) cytosolic region in a novel way. The interaction is believed to be novel because the complement of amino acids at the binding surface of the SH3 domain are atypical as well as the sequence in PIRB that is recognized. The long-term goal of this research is to provide a structural explanation for how negative signals from PIRB inhibit the maturation of neurons through HACS1. In addition to its SH3 domain, HACS1 also has a sterile alpha motif (SAM) domain that is also well known for its ability to support a wide range of protein-protein interactions. Aim 2 focuses upon discovering new partners for the HACS1 SAM domain. The identification of these partners represents an essential first step towards establishing a complete functional description of HACS1. As I have determined many SAM domain structures throughout my career, I can work with my research group to propel the most interesting partners towards high-resolution structural studies. Compared to HACS1, the organization of CASKIN2 is more complex thereby earning it the designation of a molecular scaffold, rather than an adaptor. CASKIN2, and CASKIN1 by similarity, are recognized for their roles in vesicle trafficking and creating proper connections between motor neurons and muscle cells. In Aim 3, I will determine the structural basis of how the SAM domain of CASKIN2 interacts with a known, yet poorly defined region, of the LAR receptor phosphatase. The CASKIN2/LAR partnership was selected because the SAM domain is predicted to interact with LAR in a manner that is distinct from other SAM domain structures that I have solved. The biochemical and structural insights from this aim can be extended to the Liprin class of neuronal scaffolds that also use SAM domains to bind LAR.