Structure and Function of Hetero-multimeric Glutamate Receptors

项目总结 研究的总体目标是获得完整的异质结构的高分辨率结构。 多聚体N-甲基-D-天冬氨酸受体(NMDAR)。NMDAR属于离子型谷氨酸家族 受体，它介导了哺乳动物大脑中大部分兴奋性突触传递。功能失调 NMDAR与各种神经疾病和疾病有关，包括精神分裂症、抑郁症、 阿尔茨海默氏症和帕金森氏症。NMDAR的一个独特方面是它们是强制性的异种- 由GluN1和GluN2(A-D)或GluN3(A-B)亚基组成的四聚体或更高的低聚物。开业 NMDAR离子通道需要甘氨酸与GluN1和GluN3结合，谷氨酸与GluN2结合。到目前为止， NMDAR的结构研究仅限于GluN1和GluN2的异二聚体结构 胞外结构域。因此，对于亚基和结构域是如何排列形成的，没有明确的知识 异质多聚体离子通道及其跨膜离子通道孔是如何形成的 NMDAR离子通道的高钙电导和电压依赖性镁离子通道的特性 阻止。尽管有各种技术突破，但真核生物的结晶学研究取得了成功 膜蛋白由于表达、纯化和结晶困难而受到限制。 来自样品的异质性和不稳定性。重要的是，目前还没有真核生物异源的晶体结构。 到目前为止已重组生产的多聚膜蛋白。事实上，许多离子通道， 神经学中涉及的G蛋白偶联受体、受体激酶和膜内蛋白酶 疾病以天然状态下的异源多聚体的形式存在，这表明对异源多聚体的结构研究非常必要。 多聚体膜蛋白。获得了异质多聚离子通道的第一晶体结构，并 了解NMDAR的结构-功能关系，我们将进行以下两个方面的研究 目标：目标1是使用我们的新方法生产完整的异源多聚体NMDAR蛋白，并 对均一纯化的蛋白质进行生化表征；目标2是完成对 应用尖端技术在不同配体反映不同功能状态的复合体中完整的NMDAR 膜蛋白结晶学技术和验证基于结构的功能假说 生化和电生理实验。建议的研究可望顺利完成。 以产生异质多聚体离子通道的第一晶体结构，并提供一种 了解对大脑生理学和发育至关重要的NMDAR。重要的是，结构性的 在这里获得的信息也将为开发具有治疗效果的化合物提供策略。 神经紊乱和疾病。此外，这些关于NMDAR的研究将为 异质多聚体膜蛋白的结晶学指南。

PROJECT SUMMARY 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.