Ip Hermia, Stratton Kelly, Zgurskaya Helen, Liu Jun
Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
J Biol Chem. 2003 Dec 12;278(50):50474-82. doi: 10.1074/jbc.M305152200. Epub 2003 Oct 1.
The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.
大肠杆菌的多药外排系统AcrA-AcrB-TolC可将多种药物直接从细菌细胞排至外部培养基中。外排过程的机制尚未完全明确。AcrA呈细长形，被认为跨越周质空间，协调内膜蛋白AcrB和外膜蛋白TolC的协同运作。在本研究中，我们使用定点自旋标记（SDSL）电子顺磁共振（EPR）光谱来研究溶液中AcrA的分子构象。构建了十个AcrA突变体，每个突变体都有一个丙氨酸到半胱氨酸的替换，进行纯化并用氮氧化物自旋标记物标记。对自旋标记的AcrA变体进行EPR分析表明，侧链流动性与AcrA预测的二级结构一致。我们进一步证明，酸性pH会诱导AcrA的寡聚化和构象变化，且这种结构变化是可逆的。这些结果表明，AcrA在药物外排中的作用机制类似于病毒膜融合蛋白，并且AcrA可积极介导底物的外排。
