Structural Dynamics of Multi-drug Resistance ABC Transporters

临床多药耐药在治疗细菌和真菌感染及肿瘤化疗中可 由将细胞毒性分子挤出细胞的膜包埋的外排泵的表达引起。 这些泵的一个亚类由ATP结合盒（ABC）转运蛋白、ATP动力的转运蛋白、ATP转运蛋白和ATP转运蛋白组成。 各种各样的分子。这项研究的长期目标是确定蛋白质运动， 通过ABC转运蛋白进行溶质转运的能量消耗。约5%的大肠杆菌//基因组 编码ABC转运蛋白;其中之一MsbA，转换ATP能量以翻转脂质A， 穿过内膜。它在细菌体内平衡、序列和 与人类多药转运蛋白的功能相似性结合广泛的晶体学分析 使MsbA成为临床相关ATP偶联转运的生物化学和生物药理学上易于处理的模型。 我们将使用光谱技术，以获得直接的结构和动态信息明确，关键 在脂质双层和晶体构象选择性的情况下， 晶格力这些特殊的目标将测试一种运输机制，设想一种ATP-和底物- 调节开关，从而运输伴随着二聚化和解离的循环发生， 核苷酸结合域（NBD）。自旋标记将被系统地引入到蛋白质序列中 用电子顺磁谱分析了它们的迁移率、可及性和成对邻近性 (EPR)1）重建NBD的相对运动，2）绘制构象变化， 重新定向底物结合室。该建议的一个新颖方面是使用互补的 光谱标尺，5- 80 A的距离范围，以解决晶体结构的争议方面 并为这些静态快照添加动态维度。拟议的研究将弥合目前的分歧 ABC运输机的结构和机械模型之间，并提供了一个独特的动态视角 一个重要的生化过程。除了控制药物的药代动力学特征外， 作为异种毒素的一部分，人类ABC转运蛋白在许多遗传性疾病中起致病作用， 纤维化了解它们的机制将有助于设计新的药物来克服耐药性。 化疗和遗传性疾病的治疗策略的发展。

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.