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: 7907063
• 负责人: Hassane S Mchaourab
• 金额: $ 16.53万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-31 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7907063
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Active Biological Transport; Address; Amino Acid Sequence; Architecture; Binding; Biochemical; Biological Models; Cells; Chemotherapy-Oncologic Procedure; Clinical; Couples; Crystallography; Cystic Fibrosis; Data; Development; Dimensions; Dimerization; Dissociation; Electrons; Energy Metabolism; Energy Transfer; Equilibrium; Escherichia; Fluorescence; Funding; Genome; Goals; Hereditary Disease; Homeostasis; Human; Hydrolysis; Inherited; Label; Ligands; Lipid A; Lipid Bilayers; Lipids; Lipopolysaccharides; Liposomes; Maps; Measurement; Measures; Membrane; Modeling; Molecular Conformation; Morphologic artifacts; Motion; Movement; Multi-Drug Resistance; Mycoses; Nature; Nucleotides; Pathology; Peptide Sequence Determination; Pharmaceutical Preparations; Play; Process; Proteins; Publishing; Pump; Reagent; Relative (related person); Reporter; Request for Proposals; Research; Research Personnel; Resistance; Rest; Role; Scanning; Shapes; Side; Site; Spectrum Analysis; Spin Labels; Stroke; Structure; Techniques; Temperature; Testing; Transmembrane Domain; base; chemotherapy; clinically relevant; cytotoxic; design; dimer; efflux pump; fascinate; flexibility; global environment; luminescence resonance energy transfer; multi drug transporter; novel; periplasm; programs; quantum; solute; therapeutic development; tool

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. Thespecific aims willtest a mechanism of transport that envisions anATP- 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, accessibilites 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.

临床多药在治疗细菌和真菌感染和癌症化学疗法方面的耐药性可以 由膜上包裹的外排泵表达产生的，该泵将细胞毒性分子从细胞中挤出。 这些泵的一个子类包括ATP结合盒（ABC）转运蛋白，ATP驱动的贩运者 各种分子。这项研究的长期目标是定义伴侣的蛋白质运动 ABC转运蛋白溶质易位的能量消耗。大约5％的Escherichia Co //基因组 编码ABC转运蛋白；其中之一是MSBA，将ATP能量转换为翻转脂质A，构建块 外膜的内膜。它在细菌稳态，序列和 与人类多药转运蛋白的功能相似性以及广泛的晶体学分析 使MSBA成为临床相关的ATP耦合转运的生物化学和生物物理上的模型。 我们将使用光谱技术来获取有关定义明确的关键的直接结构和动态信息 MSBA在脂质双层中的催化中间体，在没有晶体的构象中 晶格部队。第三特定的目标将使某种运输机制设想ANATP和底物 - 受调节的开关与二聚和解离的周期同时发生的开关 核苷酸结合结构域（NBD）。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分析的成对接近度 （EPR）至1）重建NBD的相对运动和2）映射构象变化 重新定位底物结合室。该提议的一个新颖方面是使用补充 具有5-80A距离范围的光谱尺尺，以解决晶体结构的有争议方面 并在这些静态快照中添加动态尺寸。拟议的研究将弥合当前鸿沟 在ABC转运蛋白的结构和机械模型之间，并提供独特的动态视角 在基本的生化重要性过程中。除了控制药物动力学特征 异种毒素，人类ABC转运蛋白在包括囊性在内的多种遗传疾病中扮演致病作用 纤维化。了解它们的机制将有助于设计新药，以克服对 化学疗法和遗传病理学治疗策略的发展。