Dynamic properties of a glutamate binding domain

描述（由申请人提供）：嗜离子性谷氨酸受体控制各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。对跨膜拓扑结构的分析表明，每个亚基都是由一系列模块组成的。结合谷氨酸的模块可以作为可溶性蛋白（S1S2结构域）在细菌中产生，其结构已经确定。AMPA受体的S1S2结构域结合激动剂和拮抗剂的亲和力与完整的受体大致相同，是研究结合结构域的一个很好的系统。在之前的授权期内，使用核磁共振光谱研究了一系列与GluR2 S1S2结合的部分激动剂的主链和侧链动力学。这些测量，结合溶液结构、新晶体结构和等温滴定量热法（ITC）的研究，揭示了激动剂和拮抗剂结合的机制，以及结合域的动力学与通道门控之间的关系。基于这些结果，动力学和通道门控之间的关系可以在更定量的水平上进行探讨，使用诱变设计来修改与激动剂功效相关的大尺度叶运动和骨干运动。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。由于谷氨酸受体被认为是二聚体的二聚体，因此激活通道的合适模型将是S1S2结构域的二聚体。该蛋白在6毫米以下为单体，但L483Y突变导致浓度低得多的二聚体蛋白。这种突变阻断了完整蛋白的脱敏，而脱敏与二聚体界面的解离有关。初步研究表明，二聚化导致激动剂结合位点的变化，目标将是确定二聚化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脱敏后的变化提供线索。最后，我们将研究变构激活剂。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。核磁共振波谱、x射线晶体学、放射性配体结合和ITC将用于确定与一系列与结合域不同相互作用的激活剂的结合机制和与受体的重要相互作用。这些研究将使用一系列生物物理技术，包括全细胞和膜片钳记录受体功能，来研究GluR2 AMPA受体上的激动剂和变构激活剂结合位点。该结果将揭示谷氨酸受体重要亚基的结构、功能和动力学的相关性，并为拮抗剂或变构激活剂的开发提供重要信息。

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.