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.

细菌和真菌感染治疗以及癌症化疗中的临床多药耐药性可以 是由膜嵌入的外排泵的表达导致的，该外排泵将细胞毒性分子挤出细胞。 这些泵的一个子类由 ATP 结合盒 (ABC) 转运蛋白、ATP 驱动的转运蛋白组成 广泛的分子范围。这项研究的长期目标是定义耦合的蛋白质运动 ABC 转运蛋白转运溶质的能量消耗。大约 5% 的大肠杆菌 Co// 基因组 ABC 转运蛋白编码；其中之一，MsbA，可转换 ATP 能量以翻转脂质 A，脂质 A 是构建模块 穿过外膜，穿过内膜。它在细菌稳态、序列和 结合广泛的晶体学分析，与人类多药转运蛋白的功能相似 使 MsbA 成为临床相关 ATP 耦合转运的生化和生物物理易处理模型。 我们将使用光谱技术来获取有关明确定义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，并且在晶体没有构象选择性的情况下 晶格力。具体目标将测试一种设想 ATP 和底物的运输机制。 调节开关，其中运输与二聚化和解离循环同时发生 核苷酸结合域（NBD）。自旋标签将被系统地引​​入蛋白质序列中 以及通过电子顺磁光谱分析它们的迁移率、可及性和成对邻近度 (EPR) 1) 重建 NBD 的相对运动，2) 绘制构象变化图 重新定向基板结合室。该提案的一个新颖之处是使用互补的 距离范围为 5-80A 的光谱尺，可解决晶体结构的争议问题 并向这些静态快照添加动态维度。拟议的研究将弥合当前的分歧 ABC 转运体的结构和机械模型之间的关系，并提供独特的动态视角 一个具有基本生化重要性的过程。除了控制药代动力学特征外 异毒素、人类 ABC 转运蛋白在许多遗传性疾病（包括囊性遗传性疾病）中发挥着致病作用 纤维化。了解它们的机制将有助于设计新药来克服耐药性 化疗和遗传性疾病治疗策略的开发。