Siderophore transport through Escherichia coli outer membrane receptor FhuA with disulfide-tethered cork and barrel domains
Siderophore transport through Escherichia coli outer membrane receptor FhuA with disulfide-tethered cork and barrel domains
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DOI：
10.1074/jbc.m506708200
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发表时间：
2005-08-26
影响因子：
4.8
通讯作者：
Coulton, JW
中科院分区：
文献类型：
--
作者：
Eisenhauer, HA;Shames, S;Coulton, JW
羟酸铁载体受体FhuA是一种依赖于tonb的大肠杆菌外膜蛋白，由一个c端22链β -桶状结构域和一个n端球状软木结构域组成。在铁载体运输到外周质过程中，FhuA软木结构域发生构象变化，允许运输通过桶管腔；或者，软木塞可以完全从桶中移开。为了探索这种变化，在假定的铁载体运输途径中，在软木结构域（L109C和Q112C）和桶状结构域（S356C和M383C）中产生了位点导向的半胱氨酸突变体。分子模型预测双半胱氨酸突变体L109C/S356C和Q112C/M383C会形成二硫键，从而拴住软木和桶状结构域。双半胱氨酸FhuA突变体在非还原条件下变性，并用硫醇特异性俄勒冈绿马来酰亚胺荧光标记。随后的SDS-PAGE分析显示了两个不同的物种：含有二硫键的FhuA和含有游离巯基的FhuA。为了确定铁载体运输途径的作用，并评估铁蛋白运输过程中软木结构域可能的重排，氧化催化剂增强了二硫键的形成。含有双半胱氨酸FhuA突变体的细胞在铁蛋白运输过程中受到氧化，表现出接近完全的二硫键形成。用二硫化物将软木栓系在木桶上后，铁蛋白的运输相当于未经处理的细胞的运输。这些结果表明，阻断假定的铁载体运输途径并不能消除铁蛋白的摄取。我们认为，在铁载体通过FhuA运输的过程中，软木结构域保留在桶内，而不是被移位。
The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain ( L109C and Q112C) and in the barrel domain ( S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.