Structures and reagents of NMDA receptors

项目摘要 本项目的目标是解开N-甲基-D-天冬氨酸中的生物活性位点的分子细节 受体（NMDAR），可开发针对精神健康相关疾病的亚型特异性试剂 以及包括抑郁症、精神病、精神分裂症、癫痫、阿尔茨海默病和中风在内的疾病， 与功能失调的NMDAR有关。NMDAR属于离子型谷氨酸受体（iGluR）家族， 介导哺乳动物脑中的大多数兴奋性突触传递。它们是异源多聚体 由GluN 1和GluN 2和/或GluN 3亚基组成的配体门控离子通道。GluN 1和GluN 3 GluN 2亚基结合包括甘氨酸和D-丝氨酸的共激动剂，而GluN 2亚基结合谷氨酸和NMDA。 每个NMDAR亚基含有氨基末端结构域（ATD）、配体结合结构域（LBD）、配体结合结构域（LBD）和配体结合结构域（LBD）。 跨膜结构域（TMD）和羧基末端结构域（CTD）。GluN 1-GluN 2 NMDAR打开它们的 甘氨酸和谷氨酸结合后的TMD离子通道。NMDAR亚型，由四种不同的GluN 2定义 亚基（A至D）或两个不同的GluN 3（A和B）与GluN 1组合，表现出不同的功能 属性和时空表达模式。在之前的资助周期中，我们解开了 GluN 1 -2A和GluN 1 -2B ATD、GluN 1 -2A和GluN 1 -2D LBD的结构，以及GluN 1 -2B的完整结构 通过利用X射线晶体学和单粒子电子低温显微镜（cryo-EM）。我们的发现提供了 对通道组装和导致激活的构象运动的基本模式的分子见解， 抑制和变构调节。尽管取得了这些进展， 仍然没有答案，包括完全未探索的含GluN 2C的NMDAR结构， 具有临床相关性的未知/未表征的变构和通道阻滞位点，以及 抗体的我们现在将进行旨在弥补这些不足的研究。目标1将决定第一个 含GluN 2C的NMDAR的结构;目的2将鉴定和表征临床上 相关化合物，包括氯胺酮、苯环己哌啶、美金刚和Glyx 13。目标3将解开 新型工程抗体的结合和功能调节机制以及自身免疫抗体 第一次这三个目标将通过获得完整NMDAR的结构信息来实现， 在存在和不存在试剂的情况下通过利用X-射线晶体学和单粒子 冷冻电镜基于结构的机制假说将主要通过电生理学来检验。成功 完成拟议的研究目标将提供对NMDAR亚型的详细了解- 特异性，不同试剂的调节机制，以及控制NMDAR亚型的新方法， 将有助于开发神经系统紊乱和疾病的治疗策略。

Project Summary The goal of this project is to unravel molecular details of pharmacologically active sites in N-methyl-D-aspartate receptors (NMDARs) with a scope to develop subtype-specific reagents against mental health-related disorders and diseases including depression, psychosis, schizophrenia, epilepsy, Alzheimer’s disease, and stroke where dysfunctional NMDARs are implicated. NMDARs belong to the ionotropic glutamate receptor (iGluR) family which mediates the majority of excitatory synaptic transmission in mammalian brains. They are hetero-multimeric ligand-gated ion channels composed of GluN1 and GluN2 and/or GluN3 subunits. The GluN1 and GluN3 subunits bind co-agonists including glycine and D-serine, whereas the GluN2 subunits bind glutamate and NMDA. Each NMDAR subunit contains an amino terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl terminal domain (CTD). The GluN1-GluN2 NMDARs open their TMD ion channels upon binding of glycine and glutamate. NMDARs subtypes, defined by four distinct GluN2 subunits (A through D) or two distinct GluN3 (A and B) in combination with GluN1, exhibit different functional properties and spatio-temporal expression patterns. In the previous grant cycles, we unraveled the fragment structures of GluN1-2A and GluN1-2B ATDs, GluN1-2A and GluN1-2D LBDs, and intact structures of GluN1-2B by utilizing x-ray crystallography and single-particle electron cryo-microscopy (cryo-EM). Our findings provided molecular insights into channel assembly and basic patterns of conformational movements leading to activation, inhibition, and allosteric regulation. Despite these advances, there are still many fundamental questions remaining unanswered, including the completely unexplored structures of the GluN2C-containing NMDARs, unknown/uncharacterized allosteric and channel blockade sites with clinical relevance, and novel regulation by antibodies. We will now conduct research aimed at fulfilling these shortfalls. Aim 1 will determine the first structure of GluN2C-containing NMDARs; Aim 2 will identify and characterize pharmacological sites of clinically relevant compounds including ketamine, phencyclidine, memantine, and Glyx13. Aim 3 will unravel the mechanism of binding and functional regulation by novel engineered antibodies, and the auto-immune antibody for the first time. These three aims will be achieved by obtaining the structural information of intact NMDAR and domain fragments in the presence and absence of reagents by utilizing x-ray crystallography and single-particle cryo-EM. Structure-based mechanistic hypotheses will be examined mainly by electrophysiology. Successful completion of the proposed research aims will provide a detailed understanding about NMDAR subtypes- specificity, the regulatory mechanism by different reagents, and novel means to control NMDAR subtypes, which will help develop therapeutic strategies for neurological disorders and diseases.