Dynamic properties of a glutamate binding domain

描述(申请人提供)：离子型谷氨酸受体控制多种正常的神经元过程，包括学习和记忆。这些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。对跨膜拓扑的分析导致了这样的认识，即每个亚单位由一系列模块组成。结合谷氨酸的模块可以在细菌中以可溶性蛋白(S1S2结构域)的形式产生，其结构已经确定。AMPA受体的S1S2结构域与激动剂和拮抗剂结合的亲和力与完整受体的亲和力大致相同，是研究结合结构域的良好系统。在之前的授予期间，用核磁共振波谱研究了一系列与GluR2 S1S2结合的部分激动剂的主链和侧链动力学。通过对溶液结构、新晶体结构和恒温滴定热法(ITC)的研究，这些测量揭示了激动剂和拮抗剂结合的机制，以及结合结构域的动力学与通道门控的关系。基于这一结果，可以在更定量的水平上探讨动力学和通道门控之间的关系，使用旨在修改与激动剂疗效相关的大规模肺叶运动和脊椎运动的突变。特别是，S1S2结构域的构象变化导致通道激活的机制将被研究。因为谷氨酸受体被认为是二聚体的二聚体，所以激活通道的合适模型应该是S1S2结构域的二聚体。该蛋白在6 mM以下为单体，但L483Y突变导致的二聚体蛋白浓度要低得多。这种突变阻止了完整蛋白质的脱敏，而脱敏与二聚体界面的解离有关。初步研究表明，二聚作用会导致激动剂结合部位的改变，目的是确定二聚作用对激动剂和拮抗剂结合的动力学和热力学的影响。二聚体和单体状态之间的差异应该为蛋白质在脱敏过程中如何变化提供线索。最后，对变构激活剂进行了研究。这些药物可以增强认知能力，目前正在对阿尔茨海默病等神经系统疾病进行测试。核磁共振光谱、X射线结晶学、放射性配基结合和ITC将被用来确定与受体的结合机制和与受体的重要相互作用，这些激活剂与结合结构域具有不同的相互作用。这些研究将使用全细胞和膜片钳记录受体功能的一系列生物物理技术来研究GluR2 AMPA受体上的激动剂和变构激动剂结合位点。这些结果将有助于揭示谷氨酸受体一个重要亚基的结构、功能和动力学之间的关系，并为开发作为拮抗剂或变构激动剂的药物提供必要的信息。 公共卫生相关性：AMPA受体介导了中枢神经系统中大部分快速兴奋性突触传递。这些受体的过度活动与中风和癫痫的病理效应有关，AMPA受体活性的增强被证明有利于提高认知能力。因此，这些受体的激动剂和激活剂在治疗上都可能是非常重要的。针对特定亚型和剪接变体的药物有可能有更多的选择性作用，副作用可能更少。这些研究的目的是了解AMPA受体(GluR2)的结构、功能和动力学，并为开发新的治疗药物提供基础。

DESCRIPTION (provided by applicant): Ionotropic glutamate receptors control a wide variety of normal neuronal processes including learning and memory. Activation of these neurotransmitter receptors is involved in a number of neurodegenerative diseases, notably stroke and epilepsy. Analysis of the transmembrane topology has led to the realization that each subunit is made of a series of modules. The module that binds glutamate can be produced in bacteria as a soluble protein (S1S2 domain) and its structure has been determined. The S1S2 domain of AMPA receptors binds agonists and antagonists with approximately the same affinity as the intact receptor and serves as an excellent system for studying the binding domain. In the previous granting period, backbone and sidechain dynamics were studied with a series of partial agonists bound to GluR2 S1S2 using NMR spectroscopy. These measurements, with studies of solution structure, new crystal structures, and isothermal titration calorimetry (ITC), have shed light on the mechanism of agonist and antagonist binding and the relationship between dynamics of the binding domain and channel gating. Based on the results, the relationship between dynamics and channel gating can be approached on a more quantitative level, using mutagenesis designed to modify large-scale lobe motions and backbone motions that correlate with agonist efficacy. In particular, the mechanism by which conformational changes in the S1S2 domain lead to channel activation will be investigated. Because glutamate receptors are thought to function as dimers of dimers, an appropriate model for the activated channel would be the dimer of the S1S2 domain. The protein is monomeric below 6 mM, but the L483Y mutation results in dimeric protein at much lower concentrations. This mutation blocks desensitization in the intact protein, and desensitization is correlated with the dissociation of the dimer interface. Preliminary studies indicate that dimerization leads to changes in the agonist binding site, and the goal will be to determine the effects of dimerization on dynamics and thermodynamics of agonist and antagonist binding. Differences between the dimeric and monomeric states should provide clues as to how the protein changes upon desensitization. Finally, allosteric activators will be studied. These drugs enhance cognition and are being tested in neurological disorders such as Alzheimer's disease. NMR spectroscopy, X-ray crystallography, radioligand binding, and ITC will be used to determine the mechanisms of binding and important interactions with the receptor for a series of activators with differing interactions with the binding domain. These studies will use a range of biophysical techniques with whole cell and patch clamp recording of receptor function to study the agonist and allosteric activator binding sites on the GluR2 AMPA receptor. The results will shed light on the correlation of structure, function and dynamics of an important glutamate receptor subunit and provide essential information for development of drugs that are antagonists or allosteric activators. PUBLIC HEALTH RELEVANCE: AMPA receptors mediate the majority of fast excitatory synaptic transmission in the central nervous system. Over-activity of these receptors has been implicated in contributing to the pathological effects of stroke and epilepsy, and enhancement of the activity of AMPA receptors has been shown to be beneficial in increasing cognition. For this reason, both agonists and activators of these receptors are likely to be very important therapeutically. Drugs targeted to specific subtypes and splice variants have the potential for more selective action with possibly fewer side effects. The goal of these studies is to understand the structure, function and dynamics of one of the most prevalent and medically relevant AMPA receptors (GluR2), and to provide the groundwork for the development of new therapeutic agents.