Dynamic properties of a glutamate binding domain

描述（由申请人提供）：嗜电性谷氨酸受体控制多种正常神经元过程，包括学习和记忆。此外，这些重要的神经递质受体的激活与许多神经退行性疾病有关，特别是中风和癫痫。因此，针对谷氨酸受体的药物将具有相当大的治疗价值。对跨膜拓扑结构的分析表明，每个亚基都由一系列模块组成，结合谷氨酸的模块可以在细菌中作为可溶性蛋白（S1S2结构域）产生。S1S2结构域结合激动剂和拮抗剂的亲和力与完整受体大致相同，是研究结合结构域的一个很好的系统。E. Gouaux及其合作者在几种激动剂和拮抗剂的存在下解决了GluR2亚基S1S2结构域的晶体结构，这为这些受体的分子理解提供了重大突破。这一建议的目的是补充已知的三维结构与动态信息从核磁共振光谱。初步研究表明，GluR2的S1S2结构域非常适合这些研究。骨架共振已经确定，结合位点上谷氨酸的初步结果提供了结合位点移动部分的图像，以及谷氨酸解离的潜在机制。在各种激动剂和拮抗剂存在的情况下，在不同温度下对主链和侧链动力学的进一步研究，将提供配体结合的能量学、与结合相关的动力学以及结合对通道功能的潜在转导的详细分子图谱。第二个目标是测量溶液中结合时的构象变化。从晶体结构可以看出，S1S2结构域是一种双叶体结构，在配体结合时关闭。然而，在类似的体系中，叶瓣闭合程度受到晶体堆积和叶瓣动力学的影响，这表明溶液中的叶瓣闭合可能与晶体中的叶瓣闭合不同。剩余偶极耦合测量将用于研究两个叶的取向，不同的配体在结合位点和不同的温度下。这将允许对溶液状态的三维结构进行外推，并提供关于结合能量学的额外信息。这些研究结果将揭示谷氨酸受体重要亚基的结合位点，并为进一步的药物开发提供重要信息。

DESCRIPTION (provided by applicant): lonotropic glutamate receptors control a wide variety of normal neuronal processes, including learning and memory. In addition, activation of these important neurotransmitter receptors is involved in a number of neurodegenerative diseases, notably stroke and epilepsy. Thus, drugs targeted toward glutamate receptors would be of considerable therapeutic value. Analysis of the transmembrane topology led to the realization that each subunit is made up of a series of modules, and the module that binds glutamate can be produced in bacteria as a soluble protein (S1S2 domain). The S1S2 domain binds agonists and antagonists with approximately the same affinity as the intact receptor and serves as an excellent system for studying the binding domain. The crystal structure of the S1S2 domain of the GluR2 subunit has been solved in the presence of several agonists and antagonists by E. Gouaux and collaborators, which provided a major breakthrough in the molecular understanding of these receptors. The purpose of this proposal is to complement the known three-dimensional structure with dynamic information from NMR spectroscopy. Preliminary studies have shown that the S1S2 domain of GluR2 is well suited to these studies. The backbone resonances have been assigned, and preliminary results with glutamate in the binding site provide a picture of the portions of the binding site that are mobile, and a potential mechanism for glutamate dissociation. Further studies of the backbone and sidechain dynamics, in the presence of various agonists and an antagonist, and at different temperatures, will provide a detailed molecular picture of the energetics of ligand binding, and the dynamics associated with binding and, potentially, of the transduction of binding to channel function. A second goal is to measure the conformational change upon binding in solution. As shown by the crystal structures, the S1S2 domain is a bilobe structure that closes upon ligand binding. However, the degree of lobe closure has been shown in similar systems to be influenced by crystal packing and lobe dynamics, suggesting that lobe closure in solution may differ from that in the crystal. Residual dipolar coupling measurements will be used to study the orientation of the two lobes, with different ligands in the binding site and at different temperatures. This should allow an extrapolation of the three-dimensional structure to the solution state and provide additional information on the energetics of binding. The results of these studies will shed light on the binding site of an important glutamate receptor subunit, and provide essential information for further drug development.