Mutant analysis of the Escherichia coli FhuA protein reveals sites of FhuA activity
Mutant analysis of the Escherichia coli FhuA protein reveals sites of FhuA activity
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DOI：
10.1128/jb.185.16.4683-4692.2003
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发表时间：
2003-08-01
影响因子：
3.2
通讯作者：
Braun, V
中科院分区：
文献类型：
--
作者：
Endriss, F;Braun, M;Braun, V
大肠杆菌的FhuA外膜蛋白主动转运铁色素、白霉素和利福霉素CGP 4832，并赋予对微菌素J25、大肠杆菌素M和pETT 1、T5和phi 80的敏感性。在FhuA晶体结构和对FhuA可能如何起作用的预测的指导下，在软木结构域（残基I至160）和P-桶结构域（残基161至714）中分离突变体。TonB盒（残基7至11）的缺失完全灭活了FhuA的所有TonB依赖性功能。通过引入的C27和C533残基之间的二硫桥将软木固定到桶的第7转，废除了铁色素运输，通过二硫键的还原恢复了铁色素运输。缺失残基24至31，包括开关螺旋（残基24至29），其在铁色素与FhuA结合时经历大的结构转变（17埃）并将FhuA的N末端（TonB盒）暴露于周质，降低了FhuA转运活性（野生型活性的79%），但赋予了对大肠杆菌素M和大肠杆菌素的完全敏感性。残基23至30的复制或残基13至20的缺失导致FhuA衍生物具有与具有残基24至31的缺失的FhuA类似的性质。然而，移码突变将位置18至21处的QSEA改变为KKAP，几乎完全消除了FhuA的大部分活性。能量偶联外膜转运蛋白中的保守残基R93和R133被认为通过与β-桶的保守残基E522和E571形成盐桥而将软木固定到β-桶。具有E522 R和E571 R突变的蛋白质是无活性的，但无活性不是由R522和R571对R93的排斥以及R571对R133的排斥引起的。软木塞中的点突变在铁色素结合后移动或不移动的位点上没有影响或仅赋予轻微降低的活性。可以得出结论，TonB框是必不可少的FhuA活性。TonB盒区域必须是灵活的，但它与软木结构域的距离可以有很大的变化。去除软木塞和桶之间的盐桥影响FhuA的结构，但不影响其功能。
The FhuA outer membrane protein of Escherichia coli actively transports ferrichrome, albomycin, and rifamycin CGP 4832, and confers sensitivity to microcin J25, colicin M, and the phages T1, T5, and phi80. Guided by the FhuA crystal structure and derived predictions on how FhuA might function, mutants were isolated in the cork domain (residues I to 160) and in the P-barrel domain (residues 161 to 714). Deletion of the TonB box (residues 7 to 11) completely inactivated all TonB-dependent functions of FhuA. Fixation of the cork to turn 7 of the barrel through a disulfide bridge between introduced C27 and C533 residues abolished ferrichrome transport, which was restored by reduction of the disulfide bond. Deletion of residues 24 to 31, including the switch helix (residues 24 to 29), which upon binding of ferrichrome to FhuA undergoes a large structural transition (17 Angstrom) and exposes the N terminus of FhuA (TonB box) to the periplasm, reduced FhuA transport activity (79% of the wild-type activity) but conferred full sensitivity to colicin M and the phages. Duplication of residues 23 to 30 or deletion of residues 13 to 20 resulted in FhuA derivatives with properties similar to those of FhuA with a deletion of residues 24 to 31. However, a frameshift mutation that changed QSEA at positions 18 to 21 to KKAP abolished almost completely most of FhuA's activities. The conserved residues R93 and R133 among energy-coupled outer membrane transporters are thought to fix the cork to the P-barrel by forming salt bridges to the conserved residues E522 and E571 of the beta-barrel. Proteins with the E522R and E571R mutations were inactive, but inactivity was not caused by repulsion of R93 by R522 and R571 and of R133 by R571. Point mutations in the cork at sites that move or do not move upon the binding of ferrichrome had no effect or conferred only slightly reduced activities. It is concluded that the TonB box is essential for FhuA activity. The TonB box region has to be flexible, but its distance from the cork domain can greatly vary. The removal of salt bridges between the cork and the barrel affects the structure but not the function of FhuA.