Killmann H, Benz R, Braun V
Universität Tübingen, Germany.
EMBO J. 1993 Aug;12(8):3007-16. doi: 10.1002/j.1460-2075.1993.tb05969.x.
The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.
FhuA受体蛋白参与通过铁载体将Fe3+进行能量偶联运输，穿过大肠杆菌的外膜。由于在外膜中尚未发现能量来源，因此推测能量是通过TonB、ExbB和ExbD蛋白的作用提供的，这些蛋白锚定在细胞质膜上。通过删除假定的细胞表面暴露环的34个氨基酸残基，FhuA从一种配体特异性转运蛋白转变为一种不依赖TonB的非特异性扩散通道。与未增加膜电导的野生型FhuA相反，FhuA缺失衍生物FhuA delta 322 - 355在黑色脂质膜中形成了稳定的通道。FhuA突变体通道的单通道电导至少比大肠杆菌外膜的一般扩散孔蛋白大3倍。有人提出，外膜中FhuA的基本结构是由β桶形成的通道。由于从第316位到356位延伸的环是FhuA活性位点的一部分，它可能控制着通道的通透性。FhuA的转运活性构象是由TonB诱导的构象变化介导的，以响应带电的细胞质膜。与在5 microM时表现出饱和的FhuA野生型细胞相反，铁载体运输到表达FhuA delta 322 - 355的细胞中的速率随着底物浓度的增加（从0.5到20 microM）呈线性增加。这意味着在野生型细胞中，铁载体通过外膜的运输是限速步骤，并且TonB、ExbB和ExbD仅在外膜运输中是必需的。
