Crucial Role of Asp408 in the Proton Translocation Pathway of Multidrug Transporter AcrB: Evidence from Site-Directed Mutagenesis and Carbodiimide Labeling
Crucial Role of Asp408 in the Proton Translocation Pathway of Multidrug Transporter AcrB: Evidence from Site-Directed Mutagenesis and Carbodiimide Labeling
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DOI：
10.1021/bi900446j
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发表时间：
2009-06-30
期刊：
影响因子：
2.9
通讯作者：
Pos, Klaas M.
中科院分区：
文献类型：
--
作者：
Seeger, Markus A.;von Ballmoos, Christoph;Pos, Klaas M.
大肠杆菌的三组分AcrA/AcrB/TolC外排系统催化质子动力驱动的多种细胞毒性化合物的挤出。内膜泵成分AcrB属于耐药细胞分裂和细胞分裂（RND）超家族，负责整个三联外排系统的药物特异性和能量转导。系统的可滴定和极性膜定位的氨基酸突变分析显示，四个残基，D407，D408，K940，和，R971，是最重要的AcrB功能。使用基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱，D408显示出特异性反应与二环己基碳二亚胺（DCCD）在pH依赖性的方式。D408的表观pK（a）为7.4，能够在生理条件下结合和释放质子。与其他二级转运蛋白相比，D408在药物存在下不受碳二亚胺修饰的保护，这支持药物和质子的空间分离的转运途径的概念。这项研究提供了一个实质性的作用，膜定位的羧酸作为一个核心元素的质子转运途径中的AcrB和其他成员的RND超家族的证据。
The three-component AcrA/AcrB/TolC efflux system of Escherichia coli catalyzes the proton motive force-driven extrusion of a variety of cytotoxic compounds. The inner membrane pump component AcrB belongs to the resistance nodulation and cell division (RND) superfamily and is responsible for drug specificity and energy transduction of the entire tripartite efflux system. Systematic mutational analysis of titratable and polar membrane-located amino acids revealed four residues, D407, D408, K940, and, R971, to be of prime importance for AcrB function. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, D408 was shown to specifically react with dicyclohexylcarbodiimide (DCCD) in a pH-dependent manner. The apparent pK(a) of D408 of 7.4 would enable binding and release of protons under physiological conditions. In contrast to other secondary transporters, D408 was not protected from carbodiimide modification in the presence of drugs, which supports the notion of spatially separated transport pathways for drugs and protons. This study provides evidence for a substantial role of membrane-located carboxylates as a central element of the proton translocation pathway in AcrB and other members of the RND superfamily.