Different Functions of MdtB and MdtC Subunits in the Heterotrimeric Efflux Transporter MdtB2C Complex of Escherichia coli
Different Functions of MdtB and MdtC Subunits in the Heterotrimeric Efflux Transporter MdtB2C Complex of Escherichia coli
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DOI：
10.1021/bi300379y
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发表时间：
2012-05-22
期刊：
影响因子：
2.9
通讯作者：
Nikaido, Hiroshi
中科院分区：
文献类型：
--
作者：
Kim, Hong-Suk;Nikaido, Hiroshi
与RND（抗性-增殖-分裂）超家族的同源三聚体转运蛋白（通常通过功能旋转机制进行广泛底物的外排转运）相反，该家族的异源三聚体成员（也进行多药外排）所利用的机制尚不清楚。我们通过对可能参与配体转运的残基进行广泛的半胱氨酸扫描诱变，研究了一种异源三聚体转运蛋白，即大肠杆菌的MdtB（2）C复合物。MdtC中的许多这样的突变强烈地降低了氯唑西林转运的水平，而MdtB中相应残基的突变不影响转运。此外，许多这样的残基在MdtC共价修饰的荧光素马来酰亚胺，这作为一个底物，并推测产生标记的残基在底物路径。相比之下，很少的残基在MdtB标记。结合先前的数据显示，质子转运通道在MdtC中的失活对转运只有适度的影响，而在MdtB中完全失活活性，这些结果表明，两个亚基，MdtB和MdtC，发挥非常不同的作用，MdtC可能参与底物结合和运输，MdtB可能诱导构象变化所需的运输通过质子转运。基于与AcrB转运蛋白的序列同源性，MdtB和MdtC的三维模型也支持这种解释。
In contrast to homotrimeric transporters of the RND (resistance-nodulation-division) superfamily, which often conduct efflux transport of a wide range of substrates by the functionally rotating mechanism, the mechanism utilized by the heterotrimeric members of this family, which also perform multidrug efflux, is unclear. We examined one heterotrimeric transporter, the MdtB(2)C complex of Escherichia coli, by an extensive cysteine scanning mutagenesis of residues likely involved in ligand transport. Many such mutations in MdtC strongly decreased the level of cloxacillin transport, whereas mutations of corresponding residues in MdtB did not affect transport. Furthermore, many such residues in MdtC were covalently modified by fluorescein maleimide, which acted as a substrate and presumably produced labeling of the residues in the substrate path. In contrast, few residues in MdtB were labeled. Together with the previous data showing that the inactivation of proton translocation channel in MdtC has an only modest effect on transport yet in MdtB totally inactivated the activity, these results suggest that the two subunits, MdtB and MdtC, play very different roles, MdtC likely involved in substrate binding and transport and MdtB presumably inducing the conformational change needed for transport through proton translocation. Three-dimensional models of MdtB and MdtC, based on sequence homology with the AcrB transporter, also support this interpretation.