In vivo reconstitution of the FhuA transport protein of Escherichia coli K-12
In vivo reconstitution of the FhuA transport protein of Escherichia coli K-12
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DOI：
10.1128/jb.185.18.5508-5518.2003
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发表时间：
2003-09-01
影响因子：
3.2
通讯作者：
Braun, V
中科院分区：
文献类型：
--
作者：
Braun, M;Endriss, F;Braun, V
大肠杆菌外膜中的FhuA蛋白主动转运铁色素和抗生素白霉素和利福霉素CGP 4832，并作为大肠杆菌素T1、T5和phi 80以及大肠杆菌素M和微菌素J25的受体。晶体结构揭示了一个β-桶与球状域，软木塞关闭通道形成的桶。软木塞的基因缺失导致了不显示FhuA活性的β桶。通过共合成分别编码的软木和β-桶结构域获得功能性FhuA，每个结构域都具有信号序列，这表明互补发生在跨细胞质膜分泌片段后。无活性的完全突变体FhuA和含有357个N-近端氨基酸残基的FhuA片段补充了单独合成的野生型β-桶以形成活性FhuA。先前声称β-桶作为转运蛋白和受体起作用是由无活性的完整FhuA和357个残基片段互补引起的。野生型软木和完整但无活性的FhuA携带软木突变，排除软木结构域的交换之间没有观察到互补。数据表明，活性FhuA是通过将单独合成的软木塞或来自完整FhuA的软木塞插入β-桶中而在细胞质外重构的，并且它们表明，在野生型FhuA中，β-桶在插入软木塞之前形成。
The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and phi80 and for colicin M and microcin J25. The crystal structure reveals a beta-barrel with a globular domain, the cork which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a beta-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the beta-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type beta-barrel to form an active FhuA. Previous claims that the beta-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the beta-barrel, and they suggest that in wild-type FhuA the beta-barrel is formed prior to the insertion of the cork.