Structural Dynamics of Drug/Proton Antiport by a Multidrug Efflux Pump

本研究旨在描述细菌多药外排泵EmrD的结构动力学特征。多药耐药现象日益成为细菌感染临床治疗的重要障碍。细菌抵抗抗生素治疗的一个关键策略是内膜中多药外排泵的表达。药物/H+反转运蛋白（DHA）是次级转运蛋白的主要促进剂超家族（MFS）的一部分，它将各种化学上不同的药物分子的外排耦合到质子在膜上的电化学梯度的耗散。对DHA家族的两个代表——乳酸乳球菌中的LmrP和大肠杆菌中的MdfA的生化研究，揭示了其底物特异性和转运机制。来自大肠杆菌的相关转运蛋白EmrD的晶体结构显示出与其他MFS转运蛋白相似的全局折叠，但其构象出乎意料。本文提出的研究将利用LmrP和MdfA的生化数据以及EmrD的结构数据，通过这一临床重要的药物外排泵家族建立药物/H+抗转运的动态机制模型。特异性目的1将评估载脂蛋白或底物结合EmrD的细胞质门的结构，以确定EmrD的晶体结构是否代表天然状态。特异性Aim 2将描述与质子结合和膜通电相关的EmrD细胞质门的构象。特异性Aim 3将映射底物与EmrD细胞质表面的结合。本研究的策略是利用定向自旋标记、电子顺磁共振（EPR）光谱和荧光光谱来报告不同条件下EmrD的结构。EPR光谱的结构数据将提供在没有洗涤剂或晶格力的情况下脂质体中EmrD的天然状态的信息。此外，这些实验将开发新的方法来监测膜蛋白在稳定质子梯度影响下的结构动力学。

This proposal seeks to characterize the structural dynamics of the bacterial multidrug efflux pump EmrD. The phenomenon of multidrug resistance is an increasingly significant obstacle to the clinical treatment of bacterial infections. One key strategy bacteria use to resist treatment with antibiotics is the expression of multidrug efflux pumps in the inner membrane. Drug/H+ antiporters (DHA), part of the major facilitator superfamily (MFS) of secondary transporters, couple the efflux of a variety of chemically dissimilar drug molecules out of the cell to the dissipation of an electrochemical gradient of protons across the membrane. Biochemical studies of two representatives of the DHA family, LmrP from Lactococcus lactis and MdfA from Escherichia coli, have shed light on the substrate specificity and the transport mechanism. A crystal structure of a related transporter, EmrD from E. coli, revealed a global fold similar to other MFS transporters but an unexpected conformation. The studies proposed here will utilize the biochemical data on LmrP and MdfA and the structural data on EmrD for the development of a dynamic, mechanistic model of drug/H+ antiport by this clinically important family of drug efflux pumps. Specific Aim 1 will evaluate the structure of the cytoplasmic gate of apo or substrate-bound EmrD to determine whether the crystal structure of EmrD represents a native state. Specific Aim 2 will characterize the conformations of the cytoplasmic gate of EmrD associated with proton binding and membrane energization. Specific Aim 3 will map substrate binding to the cytoplasmic surface of EmrD. The strategy of this investigation utilizes site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy, and fluorescence spectroscopy to report on the structure of EmrD under various conditions. Structural data from EPR spectroscopy will provide information on the native state of EmrD in liposomes without detergents or crystal lattice forces. Additionally, these experiments will develop novel methodology to monitor the structural dynamics of a membrane protein under the influence of a stable proton gradient.