Structure and Function of Hetero-multimeric Glutamate Receptors

描述（由申请方提供）：本文提出的研究的总体目标是获得完整异源多聚体N-甲基-D-天冬氨酸受体（NMDAR）的高分辨率结构。NMDAR属于离子型谷氨酸受体家族，其介导哺乳动物脑中的大多数兴奋性突触传递。功能障碍的NMDAR涉及各种神经障碍和疾病，包括精神分裂症、抑郁症、阿尔茨海默病和帕金森病。NMDAR的一个独特方面是它们是由GluN 1和GluN 2（A-D）或GluN 3（A-B）亚基组成的专性异源四聚体或更高的寡聚体。NMDAR离子通道的开放需要甘氨酸与GluN 1和GluN 3的结合以及谷氨酸与GluN 2的结合。迄今为止，NMDAR的结构研究已限于GluN 1和GluN 2胞外结构域的异源二聚体结构。因此，对于亚基和结构域如何排列以形成异源多聚体离子通道以及跨膜离子通道孔如何成形以赋予NMDAR离子通道的特定性质（包括高钙电导和电压依赖性镁阻断）没有明确的知识。尽管有各种技术突破，但由于样品异质性和不稳定性导致表达、纯化和结晶困难，真核细胞膜蛋白的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产生的真核异多聚体膜蛋白的晶体结构。神经疾病中涉及的许多离子通道、G蛋白偶联受体、受体激酶和膜内蛋白酶以天然状态的异源多聚体存在的事实表明非常需要对异源多聚体膜蛋白进行结构研究。为了获得异源多聚体离子通道的第一个晶体结构，并了解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制备完整的异源多聚体NMDAR蛋白，并对均匀纯化的蛋白进行生物化学表征;目的二是应用膜蛋白晶体学的尖端技术，完成完整NMDAR与反映不同功能状态的各种配体复合物的结构分析，并验证结构-通过生物化学和电生理学实验建立功能假说。成功完成拟议的研究预计将导致异源多聚体离子通道的第一个晶体结构，并提供对脑生理学和发育至关重要的NMDAR的机制理解。重要的是，本文获得的结构信息还将提供开发在神经障碍和疾病中具有治疗功效的化合物的策略。此外，NMDAR的这些研究将为异源多聚体膜蛋白的晶体学研究建立基本的指导方针。

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.