AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters
AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters
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DOI：
10.1128/jb.00587-15
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发表时间：
2016-01-01
影响因子：
3.2
通讯作者：
Yamaguchi, Akihito
中科院分区：
文献类型：
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作者：
Hayashi, Katsuhiko;Nakashima, Ryosuke;Yamaguchi, Akihito
大肠杆菌中的 AcrAB-TolC 系统是一种内在的 RND 型多药外排转运蛋白，其功能为内膜转运蛋白 AcrB、外膜通道 TolC 和接头蛋白 AcrA 的三联复合体。尽管该系统各组分的晶体结构已被阐明，但整个配合物的晶体结构尚未解决。现有的晶体结构表明 AcrB 和 TolC 作为三聚体发挥作用，但复合物中 AcrA 分子的数量目前仍存在争议。二硫键化学交联实验表明AcrB-AcrA-TolC的化学计量为1:1:1；另一方面，最近 AcrAB-TolC 的冷冻电子显微镜图像表明其化学计量为 1:2:1。在本研究中，我们使用各种接头构建了 1:1 固定的 AcrB-AcrA 融合蛋白。令人惊讶的是，所有 1:1 固定接头蛋白在 acrAB 缺陷条件和 acrAB acrEF 双泵敲除条件下均显示出药物输出活性，无论接头长度如何。最后，我们优化了一个较短的接头，该接头缺乏 AcrB C 末端赋予的构象自由度。这些结果表明，具有等量 AcrA 和 AcrB 的复合物足以实现药物输出功能。 重要性 RND 型多药物输出蛋白 AcrB-AcrA-TolC 复合物的结构和化学计量学仍在争论中。最近，AcrB-AcrA-TolC 复合物的电子显微镜图像已被报道，表明化学计量为 1:2:1。然而，我们在此报告，AcrB-AcrA 1:1融合蛋白在acrAB缺陷条件下以及在acrAB acrEF双缺陷条件下对药物输出具有活性，这消除了游离AcrA及其密切同源物AcrE的帮助，表明AcrB-AcrA 1:1化学计量足以满足药物输出功能。此外，AcrB-AcrA 融合蛋白无需游离 AcrA 的帮助即可发挥作用。我们认为这些结果对于考虑 AcrAB-TolC 介导的多药输出的结构和机制非常重要。
The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function.IMPORTANCEThe structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.