Dynamic properties of a glutamate binding domain

描述（由申请人提供）：离子型谷氨酸受体控制多种正常神经元过程，包括学习和记忆。这些神经递质受体的激活涉及许多神经退行性疾病，特别是中风和癫痫。跨膜拓扑结构的分析已经导致了每个亚基由一系列模块组成的认识。结合谷氨酸的模块可以在细菌中作为可溶性蛋白（S1 S2结构域）产生，并且其结构已经确定。AMPA受体的S1 S2结构域以与完整受体大致相同的亲和力结合激动剂和拮抗剂，并用作研究结合结构域的优良系统。在前一个授予期间，主链和侧链动力学进行了研究与一系列的部分激动剂结合到GluR 2 S1 S2使用NMR光谱。这些测量，与研究的溶液结构，新的晶体结构，和等温滴定量热法（ITC），揭示了激动剂和拮抗剂结合的机制和结合域的动态和通道门控之间的关系。基于这些结果，可以在更定量的水平上接近动力学和通道门控之间的关系，使用设计用于修改与激动剂功效相关的大规模叶运动和主链运动的诱变。特别是，S1 S2结构域中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是S1 S2结构域的二聚体。蛋白质在低于6 mM时是单体的，但L483 Y突变导致低得多的浓度的二聚体蛋白质。该突变阻断了完整蛋白的脱敏，并且脱敏与二聚体界面的解离相关。初步研究表明，二聚化导致激动剂结合位点的变化，目标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提供线索，蛋白质如何变化后脱敏。最后，将研究变构激活剂。这些药物可以增强认知能力，目前正在阿尔茨海默病等神经系统疾病中进行测试。NMR光谱，X-射线晶体学，放射性配体结合，和ITC将用于确定一系列激活剂与结合域的不同相互作用的结合和重要的相互作用与受体的机制。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，以研究GluR 2 AMPA受体上的激动剂和变构激活剂结合位点。这些结果将揭示一个重要的谷氨酸受体亚基的结构，功能和动力学的相关性，并为开发拮抗剂或变构激活剂的药物提供必要的信息。 公共卫生相关性：AMPA受体介导中枢神经系统中大部分快速兴奋性突触传递。这些受体的过度活性与中风和癫痫的病理作用有关，AMPA受体活性的增强已被证明有利于提高认知能力。由于这个原因，这些受体的激动剂和激活剂在治疗上可能是非常重要的。针对特定亚型和剪接变异体的药物具有更有选择性的作用，副作用可能更少。这些研究的目的是了解最普遍和医学相关的AMPA受体之一（GluR 2）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.