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

这项建议试图表征细菌多药外排泵EmRD的结构动力学。细菌多药耐药现象日益成为临床治疗细菌感染的重要障碍。细菌用来抵抗抗生素治疗的一个关键策略是在内膜表达多药外排泵。药物/氢逆向转运体(DHA)是主要的促进剂超家族(MFS)二级转运体的一部分，它将多种化学上不同的药物分子从细胞外排出，使质子在膜上的电化学梯度消散。乳酸乳球菌的LmrP和大肠杆菌的MDFA是DHA家族的两个代表，它们的生物化学研究揭示了底物的特异性和转运机制。来自大肠杆菌的相关转运蛋白EmrD的晶体结构显示了与其他MFS转运蛋白相似的全局折叠，但出现了意想不到的构象。本文提出的研究将利用LmrP和MDFA上的生化数据和EmrD上的结构数据来开发这一临床上重要的药物外排泵家族的药物/H反转运的动态机制模型。特定目的1将评估apo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自然状态。具体目标2将表征与质子结合和膜能量有关的EmrD细胞质门的构象。特异性目标3将底物结合到EmrD的细胞质表面。本研究的策略是利用定点自旋标记、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波谱的结构数据将提供有关EmrD在不含洗涤剂或晶格力的脂质体中的天然状态的信息。此外，这些实验将开发新的方法来监测在稳定的质子梯度影响下的膜蛋白质的结构动力学。

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.