Probing the Dynamics of AcrB Through Disulfide Bond Formation
Probing the Dynamics of AcrB Through Disulfide Bond Formation
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DOI：
10.1021/acsomega.0c02921
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发表时间：
2020-08
期刊：
影响因子：
4.1
通讯作者：
P. Rajapaksha;Ankit Pandeya;Yinan Wei
中科院分区：
文献类型：
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作者：
P. Rajapaksha;Ankit Pandeya;Yinan Wei
耐药-分泌-分裂（RND）超家族成员AcrA-AcrB-TolC三重外排泵是大肠杆菌多药耐药的主要原因。AcrB是外排复合物的内膜蛋白，负责在化合物运输出细胞之前识别和结合化合物。了解AcrB在药物外排过程中的功能旋转动力学是本研究的重点。为了这个目的，我们引入了六个亚基间二硫键到周质结构域的AcrB使用定点诱变研究的重要性，在亚基间接口的相对灵活性。蛋白质印迹分析显示形成二硫键连接的AcrB寡聚体，其在还原条件下被还原成单体。通过比较表达不同突变体的acrB敲除菌株的最小抑制浓度（MIC）来评价突变和二硫键形成对外排的影响。测试的双Cys突变体导致与其相应的单突变体相比相等或更高的对AcrB底物的敏感性。为了确定双突变体中活性的降低是否是由于二硫键形成对构象变化的限制，使用二硫苏糖醇（DTT）作为还原剂进行溴化乙锭蓄积试验。在两种情况下，通过DTT还原部分恢复了双Cys突变体的活性，证实了相应位置的相对运动对功能的重要性。这些发现为了解大肠杆菌AcrAB-TolC外排泵的动力学提供了新的见解。杆菌
The resistant-nodulation-division (RND) superfamily member tripartite AcrA-AcrB-TolC efflux pump is a major contributor to the multidrug resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux complex and is responsible for the recognition and binding of compounds before their transportation out of the cell. Understanding the dynamics of AcrB during functional rotation in the process of drug efflux is the focus of this study. For this purpose, we introduced six inter-subunit disulfide bonds into the periplasmic domain of AcrB using site-directed mutagenesis to study the importance of the relative flexibility at the inter-subunit interface. Western blot analysis revealed the formation of disulfide bond-linked AcrB oligomers, which were reduced into monomers under reducing conditions. The impact of mutation and formation of disulfide bond on efflux were evaluated via comparison of the minimum inhibitory concentration (MIC) of an acrB knockout strain expressing different mutants. The double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. To determine if the reduction of activity in a double mutant is due to restriction on conformational changes by the disulfide bond formation, ethidium bromide accumulation assays were conducted utilizing dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys mutants were partially restored by DTT reduction, confirming the importance of relative movement in the respective location for function. These findings provide new insights into the dynamics of the AcrAB-TolC efflux pump in E. coli.