Larsen R A, Foster-Hartnett D, McIntosh M A, Postle K
Department of Microbiology, Washington State University, Pullman 99164, USA.
J Bacteriol. 1997 May;179(10):3213-21. doi: 10.1128/jb.179.10.3213-3221.1997.
The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membrane-derived proton motive force, and an energy-transducing protein anchored in the cytoplasmic membrane, TonB. This process requires direct physical contact between the outer membrane receptor and TonB. Previous studies have identified an amino-terminally located region (termed the TonB box) conserved in all known TonB-dependent outer membrane receptors as being essential for productive energy transduction. In the present study, a mutation in the TonB box of the ferric enterochelin receptor FepA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB. Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact. This suggested that TonB residue 160, implicated in previous studies as a site of allele-specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors. This possibility was supported by the finding that TonB carboxyl-terminal truncations that retained Gln-160 were unable to participate in TonB-FepA complex formation, indicating that this site alone was not sufficient to support the physical interactions involved in energy transduction. These studies indicated that the final 48 residues of TonB were essential to this physical interaction. This region contains a putative amphipathic helix which could facilitate TonB-outer membrane interaction. Amino acid replacements at one site in this region were found to affect energy transduction but did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex. The effects of amino acid substitutions at several other TonB sites were also examined.
铁（III）-铁载体复合物和维生素B12跨大肠杆菌外膜的转运是一个主动转运过程，需要一个同源外膜受体、细胞质膜衍生的质子动力以及一个锚定在细胞质膜上的能量转导蛋白——托蛋白B（TonB）。这个过程需要外膜受体和托蛋白B之间的直接物理接触。先前的研究已经确定，在所有已知的依赖托蛋白B的外膜受体中保守的一个位于氨基末端的区域（称为托蛋白B框）对于有效的能量转导至关重要。在本研究中，铁肠螯合素受体FepA的托蛋白B框中的一个突变导致FepA与托蛋白B之间在体内可检测到的化学交联丧失。蛋白酶敏感性研究表明，这种效应是由于构象改变而非直接破坏特定的物理接触位点所致。这表明先前研究中涉及作为托蛋白B框突变体等位基因特异性抑制位点的托蛋白B残基160，对托蛋白B与外膜受体之间的物理相互作用也做出了构象上的而非直接的贡献。这一可能性得到了以下发现的支持：保留谷氨酰胺-160的托蛋白B羧基末端截短体无法参与托蛋白B-FepA复合物的形成，这表明仅该位点不足以支持能量转导中涉及的物理相互作用。这些研究表明，托蛋白B的最后48个残基对于这种物理相互作用至关重要。该区域包含一个推定的两亲性螺旋，它可能促进托蛋白B与外膜的相互作用。发现在该区域一个位点的氨基酸替换会影响能量转导，但似乎并没有极大地改变托蛋白B的构象或托蛋白B-FepA复合物的形成。还研究了托蛋白B其他几个位点的氨基酸替换的影响。
