Structural Dynamics of Multi-drug Resistance ABC Transporters

描述（由申请方提供）：细菌和真菌感染治疗和癌症化疗中的临床多药耐药性可能是由膜包埋外排泵的表达引起的，该外排泵将细胞毒性分子挤出细胞。这些泵的一个亚类由ATP结合盒（ABC）转运蛋白组成，ATP动力的大范围分子的贩运者。这项研究的长期目标是确定蛋白质运动的能量消耗耦合到溶质转运的ABC转运。大肠杆菌基因组的大约5%编码ABC转运蛋白;其中之一MsbA转导ATP能量以翻转脂质A，脂质A是外膜的构建模块，穿过内膜。它在细菌体内平衡中的关键作用，与人类多药转运蛋白的序列和功能相似性以及广泛的晶体学分析使MsbA成为临床相关ATP偶联转运的生化和生物药理学易处理模型。我们将使用光谱技术，以获得直接的结构和动态信息，定义明确，关键的催化中间体的MsbA在脂质双层和在没有构象选择性的晶格力。具体目标将测试转运机制，设想ATP和底物调节的开关，由此转运伴随核苷酸结合结构域（NBD）的二聚化和解离的循环发生。将自旋标记系统地引入蛋白质序列中，并通过电子顺磁光谱（EPR）分析其迁移率、可接近性和成对接近性，以1）重建NBD的相对运动，2）绘制重新定向底物结合室的构象变化。该提议的一个新颖方面是使用具有5- 80 A距离范围的互补光谱标尺来解决晶体结构的有争议的方面，并为这些静态快照添加动态维度。拟议的研究将弥合目前ABC转运蛋白的结构和机械模型之间的鸿沟，并提供一个独特的动态视角的基本生化重要性的过程。除了控制异种毒素的药代动力学特征外，人ABC转运蛋白在许多遗传性疾病（包括囊性纤维化）中起致病作用。了解它们的机制将有助于设计新的药物来克服对化疗的耐药性，并为遗传性疾病制定治疗策略。

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.