TM Domain Structures of Ligand-Gated Ion Channels by NMR

描述(申请人提供)：负责快速突触传递的神经递质门控受体通道的超家族在细胞间和细胞内的交流中起着至关重要的作用。这些受体包括甘氨酸、GABA-A、烟碱型乙酰胆碱和5-HT3受体，它们是多种细胞和生理功能所必需的，也是许多药物和毒物的潜在靶点。然而，由于它们的膜结合，这些蛋白质难以进行高分辨率的结构分析。它们丰富的基于序列的功能特征与缺乏高分辨率的结构和动力学信息之间的差距现在正在迅速扩大。本项目将聚焦于这个重要的受体超家族中具有代表性的成员，并在原子分辨率下研究人甘氨酸受体(Glyine Receptor，GlyR)α-1亚单位的跨膜结构。目前对跨膜通道结构的共识是由5个亚基组成的低聚物，每个亚基都有四个跨膜结构域，TM1-TM4。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模拟膜胶束和脂质双层中的高分辨率和固态核磁共振技术，将采用一种一体化的解剖-重建方法来确定GlyR的跨膜结构域结构，并逐渐增加复杂性。中心假设是跨膜结构域的二级和三级结构在很大程度上受膜环境和结构域-结构域界面侧链相互作用的影响。首席调查员和合作者取得了实质性的初步成果，以支持以下四个具体目标： 1.设计、高效表达和纯化GlyRα1亚基功能片段TM2、TMI+TM2、TM2+TM3和TMI+TM2+TM3，用于高分辨核磁共振结构和动力学研究； 2.设计TMI+TM2+TM3和TM4的节段性同位素标记策略，使单独标记的(核磁共振可见)结构域可以在整个未标记(核磁共振-不可见)TM组装的背景下进行研究； 3.在模拟膜的环境中，使用高分辨率核磁共振对跨膜结构域进行结构和动力学表征； 4.用固体核磁共振确定各TM节段之间以及相对于膜的空间方向。 拟议研究的每一个方面的可行性都得到了测试和证明。随着最近相关烟碱型乙酰胆碱受体的4-A分辨率EM结构的出现，本研究将产生原子分辨率的结构数据，这些结构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，为未来合理设计对这些受体相关疾病具有高度特异性的新的治疗药物提供高分辨率结构基础。

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.