Structural Dynamics of Multi-drug Resistance ABC Transporters

描述（由申请人提供）：在治疗细菌和真菌感染以及癌症化疗中，临床多药耐药可能是由膜嵌入外排泵的表达引起的，该外排泵将细胞毒性分子挤出细胞。这些泵的一个亚类包括ATP结合盒（ABC）转运体，ATP驱动的各种分子的贩运者。本研究的长期目标是定义蛋白质运动，将能量消耗与ABC转运蛋白的溶质转运结合起来。大约5%的埃希氏菌基因组编码ABC转运蛋白；其中之一，MsbA，转导ATP能量翻转脂质A，脂质A是外膜的组成部分，穿过内膜。MsbA在细菌稳态中的关键作用，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物理模型。我们将使用光谱学技术，在晶格力没有构象选择性的情况下，获得脂质双层中MsbA的明确定义的关键催化中间体的直接结构和动态信息。具体目标将测试一种转运机制，该机制设想ATP和底物调节的开关，其中转运与核苷酸结合域（nbd）的二聚化和解离循环同时发生。自旋标签将系统地引入蛋白质序列，并通过电子顺磁波谱（EPR）分析它们的迁移性、可及性和成对接近性，以1)重建nbd的相对运动，2)绘制重新定位底物结合室的构象变化。该方案的一个新颖之处是使用5-80A距离范围内的互补光谱尺来解决晶体结构中有争议的方面，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制模型之间的鸿沟，并为基本生化重要性的过程提供独特的动态视角。除了控制异种毒素的药物动力学外，人类ABC转运蛋白在包括囊性纤维化在内的许多遗传疾病中起致病作用。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，并为遗传病理制定治疗策略。

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.