Structural Dynamics of Multi-drug Resistance ABC Transporters

多药耐药ABC转运蛋白的结构动力学

• 批准号: 7261751
• 负责人: Hassane S Mchaourab
• 金额: $ 3.2万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7261751
• 关键词: Escherichia coli; adenosine triphosphate; bacterial proteins; bioimaging /biomedical imaging; conformation; electron spin resonance spectroscopy; fluorescent dye /probe; lipid bilayer membrane; lipid metabolism; membrane activity; membrane model; membrane transport proteins; molecular /cellular imaging; multidrug resistance; protein structure function

DESCRIPTION (provided by applicant): Clinical multidrug resistance in the treatment of bacterial and fungal infections and cancer chemotherapy can result from expression of membrane-embedded efflux pumps that extrude cytotoxic molecules out of the cell. A subclass of these pumps consists of ATP binding cassette (ABC) transporters, ATP-powered traffickers of a wide range of molecules. The long term goal of this research is to define the protein motion that couples energy expenditure to solute translocation by ABC transporters. Roughly 5% of the Escherichia Co// genome encodes for ABC transporters; one of which, MsbA, transduces ATP energy to flip lipid A, the building block of the outer membrane, across the inner membrane. Its critical role in bacterial homeostasis, sequence and functional similarity to human multidrug transporters in conjunction with extensive crystallographic analysis make MsbA a biochemically and biophysically tractable model of clinically relevant ATP-coupled transport. We will use spectroscopic techniques to obtain direct structural and dynamic information on well-defined, key catalytic intermediates of MsbA in lipid bilayers and in the absence of conformational selectivity by crystal lattice forces. The specific aims will test a mechanism of transport that envisions an ATP- and substrate- regulated switch whereby transport occurs concomitantly with cycles of dimerization and dissociation of the nucleotide binding domains (NBDs). Spin labels will be systematically introduced into the protein sequence and their mobilities, accessibilities and pairwise proximities analyzed by electron paramagnetic spectroscopy (EPR) to 1) reconstruct the relative movements of the NBDs and 2) map conformational changes that reorient the substrate binding chamber. A novel aspect of this proposal is the use of complementary spectroscopic rulers with a 5-80A distance range to address controversial aspects of the crystal structures and add a dynamic dimension to these static snapshots. The proposed studies will bridge the current divide between structural and mechanistic models of ABC transporters and provide a unique dynamic perspective on a process of fundamental biochemical importance. In addition to controlling the pharmokinetic profile of xenotoxins, human ABC transporters play causative roles in a number of genetic disorders including cystic fibrosis. Understanding their mechanisms will aid in the design of new drugs to overcome resistance to chemotherapy and the development of therapeutic strategies for the inherited pathologies.

描述（由申请人提供）：细菌和真菌感染治疗的临床多药耐药性以及癌症化学疗法可能是由于膜上包含的外排泵的表达导致的，从而将细胞毒性分子从细胞中挤出。这些泵的子类由ATP结合盒（ABC）转运蛋白，ATP驱动的贩运者组成，具有广泛的分子。这项研究的长期目标是定义蛋白质运动，使能量消耗伴随着ABC转运蛋白溶质的易位。大约5％的Escherichia Co //基因组编码ABC转运蛋白；其中之一是MSBA将ATP能量转换为将脂质A（外膜的构建块）翻转，穿过内膜。它在细菌稳态，序列和功能相似性中与人类多药物转运蛋白的关键作用与广泛的晶体学分析，使MSBA成为生物化学和生物物理上可与临床相关的ATP耦合转运的模型。我们将使用光谱技术来获取有关脂质双层中MSBA的明确定义的关键催化中间体的直接结构和动态信息，并且在没有晶体晶格力的构象选择性的情况下。具体目的将测试一种运输机制，该机制设想了ATP和底物调控的开关，该开关与二聚体的循环和核苷酸结合结构域（NBD）的离解相关。自旋标签将系统地引入蛋白质序列及其迁移率，可访问性和通过电子顺磁光谱（EPR）分析的成对接近度（EPR）至1）重建NBDS的相对运动和2）映射构象变化，以重新定位底物结合室。该建议的一个新方面是使用具有5-80A距离范围的互补光谱统治者来解决晶体结构的有争议方面，并为这些静态快照添加了动态维度。拟议的研究将弥合ABC转运蛋白的结构和机械模型之间的当前鸿沟，并就基本生化重要性过程提供独特的动态观点。除了控制异种毒素的药物动力学特征外，人ABC转运蛋白在包括囊性纤维化在内的许多遗传疾病中都起着致病作用。了解它们的机制将有助于设计新药，以克服对化学疗法的抗药性和遗传病理的治疗策略的发展。