Structural Basis of Allosteric Regulation in the NMDA Receptor

 描述（由申请人提供）：大脑中的大多数突触传递依赖于离子型谷氨酸受体。这类离子通道由四个亚家族组成，其中，N-甲基-D-天冬氨酸受体（NMDAR）对于长时程增强和学习至关重要，并且它们的失调与阿尔茨海默病、中风和许多神经系统疾病有关。NMDAR组装为专性异源四聚体，每个亚基由四个模块结构域组成：细胞外氨基末端结构域（ATD）和配体结合结构域（LBD）、形成离子通道的跨膜结构域（TMD）和非结构化细胞内羧基末端结构域（CTD）。许多因素影响NMDAR活性，包括pH，锌和镁结合，以及蛋白质-蛋白质相互作用，在广泛分布于蛋白质的位点，但这些相互作用如何转化为差异通道活性仍然是未知的。该项目旨在通过X射线晶体学，生物物理学和生物化学方法确定NMDAR活性变构调节的结构基础，主要目的有两个：1）首先，我将解决与一系列与拮抗剂Ifenprodil同源的小分子结合的NMDAR ATD的X射线晶体结构，其具有相当大的脱靶效应，但其新的变体已显示仅在缺血事件如中风期间发现的低pH环境中结合NMDAR。2)在我的第二个目标中，我将使含有ATD、LBD和TMD与激动剂精胺复合的完整受体结晶。有趣的是，该化合物似乎在ATD/LBD界面结合，并特异性地增强NMDAR剪接变体的子集;通过解决和比较精胺敏感和精胺不敏感受体的结构，我将确定配体与胞外结构域的结合如何变构改变TMD的活性。古川实验室在NMDAR方面拥有丰富的经验，我的位置独特，可以通过使用我们优化的哺乳动物表达系统、先进的大分子结晶技术（包括生物立方相方法）以及量热和电生理验证设备来回答有关NMDA受体调节的基本问题。这项研究将使我在电生理学和大型膜蛋白组装体的结构生物学的多表达系统和现代方法方面获得丰富的经验，并通过定义NMDAR的变构调节，我的工作有可能为开发一系列神经系统疾病和疾病的新型，高度靶向的治疗方法铺平道路。总之，这个项目的培训和科学成就将为我在这个领域的独立研究做好准备。

 DESCRIPTION (provided by applicant): The majority of synaptic transmission in the brain relies upon the ionotropic glutamate receptors. This class of ion channels is comprised of four subfamilies, and of these, the N-methyl-D-aspartate receptors (NMDARs) are critical for long-term potentiation and learning and their misregulation has been implicated in Alzheimer's disease, stroke, and a number of neurological disorders. The NMDAR assembles as an obligate heterotetramer with each subunit consisting of four modular domains: an extracellular Amino Terminal Domain (ATD) and Ligand Binding Domain (LBD), a transmembrane domain (TMD) which forms the ion channel, and an unstructured intracellular Carboxyl Terminal Domain (CTD). Numerous factors influence NMDAR activity, including pH, zinc & magnesium binding, and protein-protein interactions, at sites widely distributed across the protein, yet how these interactions are translated into differential channel activity remains largely unknown. This projec seeks to define the structural basis for the allosteric regulation of NMDAR activity through X-ray crystallographic, biophysical, and biochemical methods with two primary aims: 1) first, I will solve the X-ray crystal structure of the NMDAR ATD bound to a series of small molecules homologous to the antagonist Ifenprodil, which has considerable off-target effects but novel variations of which have been shown to bind the NMDAR only in the low-pH environments found during ischemic events such as stroke. 2) In my second aim, I will crystallize the intact receptor containing the ATD, LBD, and TMD in complex with the agonist spermine. Interestingly, this compound appears to bind at the ATD/LBD interface and specifically potentiates a subset of NMDAR splice variants; by solving and comparing the structures of the spermine-sensitive and spermine- insensitive receptors, I will determine how ligand binding to the extracellular domains allosterically alters the activity of the TMD. The Furukawa laboratory has extensive experience with the NMDAR, and I am uniquely situated to answer fundamental questions regarding NMDA receptor regulation through the use of our optimized mammalian expression system, advanced macromolecular crystallization techniques including lipidic cubic phase methods, and calorimetric and electrophysiological validation equipment. This research will allow me to gain tremendous experience in multiple expression systems and modern methods in electrophysiology and the structural biology of large membrane protein assemblies, and by defining the allosteric regulation of the NMDAR, my work has the potential to pave the way for the development of novel, highly-targeted therapeutics in a range of neurological disorders and diseases. Together, the training and the scientific achievements of this project will prepare me for research independence in this field.