Endriss Franziska, Braun Michael, Killmann Helmut, Braun Volkmar
Mikrobiologie/Membranphysiologie, Universität Tübingen, D-72076 Tübingen, Germany.
J Bacteriol. 2003 Aug;185(16):4683-92. doi: 10.1128/JB.185.16.4683-4692.2003.
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 1 to 160) and in the beta-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 A) 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 beta-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.
大肠杆菌的FhuA外膜蛋白可主动转运铁载体、白霉素和利福霉素CGP 4832，并赋予细胞对微菌素J25、大肠杆菌素M以及噬菌体T1、T5和phi80的敏感性。根据FhuA晶体结构以及关于FhuA可能如何发挥功能的推导预测，在塞子结构域（第1至160位氨基酸残基）和β桶结构域（第161至714位氨基酸残基）中分离出了突变体。缺失TonB框（第7至11位氨基酸残基）会使FhuA的所有TonB依赖性功能完全失活。通过在引入的C27和C533残基之间形成二硫键，将塞子固定到桶的第7圈，从而消除了铁载体的转运，而二硫键的还原可恢复铁载体的转运。缺失包括开关螺旋（第24至29位氨基酸残基）在内的第24至31位氨基酸残基，在铁载体与FhuA结合时，开关螺旋会发生较大的结构转变（17 Å），并将FhuA的N端（TonB框）暴露于周质，这降低了FhuA的转运活性（为野生型活性的79%），但赋予了对大肠杆菌素M和噬菌体的完全敏感性。重复第23至30位氨基酸残基或缺失第13至20位氨基酸残基会产生与缺失第24至31位氨基酸残基的FhuA具有相似特性的FhuA衍生物。然而，一个移码突变将第18至21位的QSEA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保守的R93和R133残基被认为通过与β桶的保守残基E522和E571形成盐桥，将塞子固定到β桶上。具有E522R和E571R突变的蛋白质无活性，但无活性并非由R522和R571对R93的排斥以及R571对R133的排斥所致。在塞子上，铁载体结合时移动或不移动的位点处的点突变没有影响，或仅使活性略有降低。得出的结论是，TonB框对FhuA活性至关重要。TonB框区域必须具有灵活性，但其与塞子结构域的距离可能有很大差异。去除塞子和桶之间的盐桥会影响FhuA的结构，但不影响其功能。
