Studies on colicin B translocation: FepA is gated by TonB
Studies on colicin B translocation: FepA is gated by TonB
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DOI：
10.1111/j.1365-2958.2007.05808.x
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发表时间：
2007-07-01
影响因子：
3.6
通讯作者：
Postle, Kathleen
中科院分区：
文献类型：
--
作者：
Devanathan, Surendranathan;Postle, Kathleen
大肠杆菌素B是一种55 kDa的哑铃形蛋白毒素，其使用TonB系统（外膜转运蛋白FepA和三种细胞质膜蛋白TonB/ExbB/ExbD）进入并杀死大肠杆菌。FepA是一个22链的β-桶，其内腔填充有一个氨基末端球状结构域，该结构域含有一个N-末端保守区，称为TonB盒，TonB与之结合。为了研究大肠杆菌素B跨外膜转运的机制，我们在FepA的球状结构域中设计了半胱氨酸（Cys）取代。大肠杆菌素B导致所有Cys取代的生物素马来酰亚胺标记的暴露增加，但程度不同，所有增加都需要TonB以及FepA TonB盒。由于从T1 3到T51，Cys残基暴露量的大幅增加，我们得出结论，大肠杆菌素B通过FepA的内腔移位，而不是沿着脂质-桶界面或通过另一种蛋白质。部分FepA球状结构域（残基V91-V142）证明相对难标记，表明相关的Cys残基被一种未知的蛋白质所隔离，或者FepA球状结构域的一个重要部分保留在桶内，需要伴随的大肠杆菌素B在其易位过程中的构象重排。出乎意料的是，TonB也是大肠杆菌素诱导的FepA TonB盒暴露所必需的，这表明TonB在与TonB盒相互作用之前在不同的位点结合FepA。
Colicin B is a 55 kDa dumbbell-shaped protein toxin that uses the TonB system (outer membrane transporter, FepA, and three cytoplasmic membrane proteins TonB/ExbB/ExbD) to enter and kill Escherichia coli. FepA is a 22-stranded beta-barrel with its lumen filled by an amino-terminal globular domain containing an N-terminal semiconserved region, known as the TonB box, to which TonB binds. To investigate the mechanism of colicin B translocation across the outer membrane, we engineered cysteine (Cys) substitutions in the globular domain of FepA. Colicin B caused increased exposure to biotin maleimide labelling of all Cys substitutions, but to different degrees, with TonB as well as the FepA TonB box required for all increases. Because of the large increases in exposure for Cys residues from T1 3 to T51, we conclude that colicin B is translocated through the lumen of FepA, rather than along the lipid-barrel interface or through another protein. Part of the FepA globular domain (residues V91-V142) proved relatively refractory to labelling, indicating either that the relevant Cys residues were sequestered by an unknown protein or that a significant portion of the FepA globular domain remained inside the barrel, requiring concomitant conformational rearrangement of colicin B during its translocation. Unexpectedly, TonB was also required for colicin-induced exposure of the FepA TonB box, suggesting that TonB binds FepA at a different site prior to interaction with the TonB box.