Structure and Function of Hetero-multimeric Glutamate Receptors

描述（由申请人提供）：本研究的总体目标是获得完整的异多聚n -甲基- d -天冬氨酸受体（NMDARs）的高分辨率结构。NMDARs属于嗜离子性谷氨酸受体家族，在哺乳动物大脑中介导大部分兴奋性突触传递。功能失调的NMDARs与多种神经系统疾病有关，包括精神分裂症、抑郁症、阿尔茨海默病和帕金森病。NMDARs的一个独特之处在于它们是由GluN1和GluN2 （A- d）或GluN3 （A- b）亚基组成的强制性异四聚体或更高的低聚物。NMDAR离子通道的打开需要甘氨酸与GluN1和GluN3以及谷氨酸与GluN2结合。迄今为止，NMDARs的结构研究仅限于GluN1和GluN2胞外结构域的异二聚体结构。因此，对于亚基和结构域是如何排列形成异质多聚离子通道的，以及跨膜离子通道孔是如何形成的，以赋予NMDAR离子通道的特定性质，包括高钙电导和电压依赖的镁块，目前还没有明确的知识。尽管有各种各样的技术突破，但由于样品的异质性和不稳定性，在表达、纯化和结晶方面存在困难，因此真核膜蛋白晶体学研究的成功受到限制。重要的是，迄今为止还没有重组生产的真核异多聚膜蛋白的晶体结构。许多离子通道、G蛋白偶联受体、受体激酶和与神经系统疾病有关的膜内蛋白酶在天然状态下作为异多聚体存在，这一事实表明了对异多聚膜蛋白结构研究的巨大需求。为了获得异多聚离子通道的第一个晶体结构，了解NMDAR的结构-功能关系，我们将进行以下两个目标的研究：目标1是使用我们的新方法生产完整的异多聚NMDAR蛋白，并对均质纯化的蛋白进行生化表征；目的2：利用膜蛋白晶体学的前沿技术，完成不同配体复合物中完整NMDARs的结构分析，并通过生化和电生理实验验证基于结构的功能假设。这项研究的成功完成有望产生异质多聚离子通道的第一个晶体结构，并提供对脑生理和发育至关重要的NMDARs的机制理解。重要的是，这里获得的结构信息也将为开发具有神经系统疾病治疗功效的化合物提供策略。此外，这些NMDARs的研究将为异质多聚膜蛋白的晶体学研究提供基本的指导。

DESCRIPTION (provided by applicant): The overall goal of the research studies proposed here is to obtain high-resolution structures of intact hetero- multimeric N-methyl-D-aspartate receptors (NMDARs). NMDARs belong to the family of ionotropic glutamate receptors, which mediate the majority of excitatory synaptic transmission in mammalian brains. Dysfunctional NMDARs are implicated in various neurological disorders and diseases including schizophrenia, depression, Alzheimer's disease, and Parkinson's disease. A unique aspect of NMDARs is that they are obligatory hetero- tetramers or higher oligomers composed of GluN1 and GluN2 (A-D) or GluN3 (A-B) subunits. Opening of NMDAR ion channels requires binding of glycine to GluN1 and GluN3 and glutamate to GluN2. To date, structural studies of NMDARs have been limited to the hetero-dimeric structures of the GluN1 and GluN2 extracellular domains. Thus, there is no clear knowledge on how subunits and domains are arranged to form hetero-multimeric ion channels and how transmembrane ion channel pores are shaped to confer specific properties of NMDAR ion channels including high calcium conductance and voltage-dependent magnesium block. Despite various technological breakthroughs, success in crystallographic studies on eukaryotic membrane proteins has been limited due to difficulties in expression, purification, and crystallization stemming from sample heterogeneity and instability. Importantly, there has been no crystal structure of eukaryotic hetero- multimeric membrane proteins that are recombinantly produced to date. The fact that numerous ion channels, G protein-coupled receptors, receptor kinases, and intramembrane proteases implicated in neurological diseases exist as hetero-multimers in native states points to the great need for structural studies on hetero- multimeric membrane proteins. To obtain the first crystal structure of hetero-multimeric ion channels and to understand the structure-function relationship of NMDARs, we will conduct research with the following two aims: Aim 1 is to produce intact hetero-multimeric NMDAR proteins using our novel methodology and to biochemical characterize the homogeneously purified proteins; and Aim 2 is to complete structural analysis of intact NMDARs in complex with various ligands reflecting different functional states by applying cutting-edge techniques in membrane protein crystallography and validate structure-based functional hypotheses by biochemical and electrophysiological experiments. Successful completion of the proposed studies is expected to result in the first crystal structure of a hetero-multimeric ion channel and to provide a mechanistic understanding of NMDARs that are critical in brain physiology and development. Importantly, the structural information obtained here will also provide strategies to develop compounds with therapeutic efficacy in neurological disorders and diseases. Furthermore, these studies on NMDARs will establish fundamental guidelines for crystallography on hetero-multimeric membrane proteins.