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

该建议旨在表征细菌多药外排泵EmrD的结构动力学。多药耐药现象是细菌感染临床治疗的一个日益显著的障碍。细菌抵抗抗生素治疗的一个关键策略是在内膜中表达多药外排泵。药物/H+反向转运蛋白（DHA）是次级转运蛋白主要易化剂超家族（MFS）的一部分，其将多种化学性质不同的药物分子流出细胞与质子跨膜的电化学梯度耗散偶联。DHA家族的两个代表，乳酸乳球菌的LmrP和大肠杆菌的MdfA的生化研究，揭示了底物特异性和运输机制。一个相关的转运蛋白，EmrD从E。大肠杆菌，揭示了一个全球折叠类似于其他MFS转运蛋白，但一个意想不到的构象。本文提出的研究将利用LmrP和MdfA的生化数据和EmrD的结构数据，通过这个临床重要的药物外排泵家族开发药物/H+反向转运的动态机制模型。具体目标1将评价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。特定目标2将表征与质子结合和膜结合相关的EmrD细胞质门的构象。特异性Aim 3将映射EmrD细胞质表面的底物结合。本研究的策略利用定点自旋标记，电子顺磁共振（EPR）光谱和荧光光谱报告在各种条件下的EmrD的结构。来自EPR光谱的结构数据将提供关于没有洗涤剂或晶格力的脂质体中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的结构动力学的影响下，一个稳定的质子梯度。

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.