TM Domain Structures of Ligand-Gated Ion Channels by NMR

描述（由申请人提供）：负责快速突触传递的神经递质门控受体通道超家族在细胞间和细胞内通讯中起着至关重要的作用。这些受体，包括甘氨酸、GABA-A、烟碱乙酰胆碱和5-HT 3受体，对于各种细胞和生理功能是必需的，并且是许多药理学和毒理学试剂的潜在靶标。然而，由于它们的膜缔合，这些蛋白质难以进行高分辨率结构分析。基于序列的功能表征的丰富性与高分辨率结构和动力学信息的缺乏之间的差距正在迅速扩大。本项目将重点关注这一重要受体超家族的代表性成员，并以原子分辨率研究人甘氨酸受体（GlyR）α-1亚基的跨膜结构域结构。跨膜通道结构的当前共识是5个亚基的寡聚体，每个亚基具有四个跨膜结构域，TM 1-TM 4。通过结合新的蛋白质表达系统，节段性同位素标记，以及最先进的高分辨率和固态NMR在膜模拟胶束和脂质双层，一个综合的解剖重建的方法将被用来确定跨膜结构域结构的GlyR逐渐增加的复杂性。中心假设是，跨膜结构域的二级和三级结构在很大程度上取决于膜环境和结构域-结构域界面侧链相互作用。主要研究者和合作者已经取得了实质性的初步成果，以支持以下四个具体目标： 1.工程化、过表达和纯化GlyR α 1亚基的功能性TM 2、TMI+ TM 2、TM 2 + TM 3和TMI+ TM 2 + TM 3片段，用于通过高分辨率NMR进行结构和动力学测量; 2.设计在TM 3和TM 4之间用人工连接物对TMI+ TM 2 + TM 3和TM 4进行片段同位素标记的策略，使得单独标记的（NMR可见）结构域可以在整个未标记的（NMR不可见）TM组装体的背景下进行研究; 3.使用高分辨率核磁共振技术对拟膜环境中的跨膜结构域进行结构和动力学表征; 4.通过固态NMR确定单个TM片段相对于彼此以及相对于膜的空间取向。 建议研究的每一个方面的可行性都得到了测试和证明。随着相关烟碱乙酰胆碱受体的4-A分辨率EM结构的最近可用性，本研究将生成具有原子分辨率的结构数据，这些数据可以作为未来探索和预测GlyR和同一超家族中其他受体的跨膜通道结构的模板，为将来合理设计对这些受体相关疾病具有高度特异性的新治疗剂提供了高分辨率的结构基础。

DESCRIPTION (provided by applicant): The superfamily of neurotransmitter-gated receptor channels responsible for fast synaptic transmission plays a crucial role in inter- and intra-cellular communication. These receptors, including glycine, GABA-A, nicotinic acetylcholine, and 5-HT3 receptors, are essential for various cellular and physiological functions and are the potential targets of many pharmacological and toxicological agents. Because of their membrane association, however, these proteins are refractory to high-resolution structural analyses. The gap is now rapidly widening between the abundance of their sequence-based functional characterizations and the lack of high-resolution structural and dynamical information. This project will focus on a representative member of this important superfamily of receptors and study the transmembrane domain structures of human glycine receptor (GlyR) alpha-1 subunit at atomic resolution. The current consensus of the transmembrane channel architecture is an oligomer of 5 subunits, each of which has four transmembrane domains, TM1-TM4. By combining the new protein expression systems, the segmental isotopic labeling, and the state-of-the art high-resolution and solid-state NMR in membrane-mimetic micelles and lipid bilayers, an integrated dissecting-rebuilding approach will be employed to determine the transmembrane domain structures of GlyR with progressively increasing complexity. The central hypothesis is that the secondary and tertiary structures of the transmembrane domains are largely governed by the membranous environment and by the domain-domain interfacial side-chain interactions. Substantive preliminary results have been obtained by the Principal Investigator and collaborators to support the following four specific aims: 1. To engineer, over-express, and purify functional TM2, TMI+TM2, TM2+TM3, and TMI+TM2+TM3 segments of GlyR alpha1 subunit for structural and dynamical measurements by high-resolution NMR; 2. To devise strategies for segmental isotopic labeling of TMI+TM2+TM3 and TM4 with an artificial linker between TM3 and TM4, so that individually labeled (NMR visible) domain can be studied in the context of the whole unlabeled (NMR-invisible) TM assembly; 3. To use high-resolution NMR for structure and dynamics characterization of the transmembrane domains in membrane-mimetic environments; and 4. To determine the spatial orientation of individual TM segments relative to each other and to the membrane by solid-state NMR. The feasibility of every aspect of the proposed studies has been test and proven. With the very recent availability of a 4-A resolution EM structure of the related nicotinic acetylcholine receptor, the present study will generate structural data with atomic resolution that can serve as templates for the future exploration and prediction of the transmembrane channel architecture for GlyR and other receptors in the same superfamily, providing a high-resolution structural basis for future rational design of new therapeutic agents that are highly specific for the diseases related to these receptors.