TM Domain Structures of Ligand-Gated Ion Channels by NMR

通过 NMR 测定配体门控离子通道的 TM 域结构

• 批准号: 6933926
• 负责人: YAN XU
• 金额: $ 26.73万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6933926
• 关键词: affinity chromatography; biomimetics; chimeric proteins; circular dichroism; esterification; glycine receptors; high performance liquid chromatography; isomerase; molecular dynamics; nuclear magnetic resonance spectroscopy; oxysteroid; protein purification; protein structure; structural biology

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.

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信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-HT3受体，对各种细胞和生理功能至关重要，是许多药理学和毒理学试剂的潜在靶点。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。目前，丰富的基于序列的功能表征与缺乏高分辨率的结构和动态信息之间的差距正在迅速扩大。本项目将以甘氨酸受体(GlyR) α -1亚基的跨膜结构为研究对象，开展甘氨酸受体(GlyR) α -1亚基跨膜结构的原子分辨率研究。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4个跨膜结构域TM1-TM4。通过结合新的蛋白质表达系统，片段同位素标记，以及在膜模拟胶束和脂质双层中最先进的高分辨率和固态核磁共振，将采用集成的解剖重建方法来确定GlyR的跨膜结构域结构，其复杂性逐渐增加。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支配。主要研究者和合作者已经取得了实质性的初步结果，以支持以下四个具体目标：