Structural Dynamics of Multi-drug Resistance ABC Transporters

描述(申请人提供)：治疗细菌和真菌感染和癌症化疗的临床多药耐药可由膜包埋外排泵的表达引起，这种外排泵可将细胞毒分子挤出细胞。这些泵的一个子类包括三磷酸腺苷结合盒(ABC)转运体，即由三磷酸腺苷提供动力的各种分子的转运体。这项研究的长期目标是定义结合能量消耗的蛋白质运动来解决ABC转运蛋白的转运问题。大约5%的大肠杆菌Co//基因组编码ABC转运蛋白；其中一种是MSBA，它传递ATP能量来翻转脂质A，即外膜的构建块，穿过内膜。它在细菌动态平衡、序列和功能上与人类多药转运蛋白的相似性，加上广泛的结晶学分析，使MSBA成为临床上相关的ATP偶联转运蛋白的生物化学和生物物理模型。我们将使用光谱技术，在没有通过晶格力进行构象选择性的情况下，在脂质双层中获得关于MSBA的定义明确的关键催化中间体的直接结构和动态信息。特定的目标将测试一种运输机制，该机制设想了一种ATP和底物调节的开关，由此运输伴随着核苷酸结合域(NBD)的二聚化和解离循环而发生。自旋标记将被系统地引入到蛋白质序列中，并通过电子顺磁谱(EPR)分析它们的迁移率、可达性和成对邻近程度，以1)重建NBD的相对运动，2)映射改变底物结合室方向的构象变化。这一提议的一个新颖方面是使用具有5-80A距离范围的互补光谱尺子来解决晶体结构的有争议的方面，并为这些静态快照增加动态维度。拟议的研究将弥合目前ABC转运蛋白结构模型和机制模型之间的分歧，并为基本生化重要性的过程提供独特的动态视角。除了控制外源毒素的药动学特征外，人类ABC转运蛋白在包括囊性纤维化在内的许多遗传性疾病中发挥着致病作用。了解它们的机制将有助于设计克服化疗耐药性的新药，并开发针对遗传性病理的治疗策略。

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.