Larsen R A, Thomas M G, Wood G E, Postle K
Department of Microbiology, Washington State University, Pullman 99164.
Mol Microbiol. 1994 Aug;13(4):627-40. doi: 10.1111/j.1365-2958.1994.tb00457.x.
Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the aminoterminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The delta V17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonB delta V17. Molecular modeling suggested that the delta V17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonB delta V17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the delta V17 mutation interrupted productive TonB-ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonB delta V17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonB delta V17 export. Unlike the tonB delta V17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonB delta V17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (approximately 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.
维生素B12和铁（III）-铁载体复合物跨大肠杆菌外膜的主动运输似乎取决于TonB蛋白将细胞质膜产生的质子动力与外膜受体偶联的能力。辅助蛋白ExbB支持TonB发挥这一作用，它除了能稳定TonB使其免受内源性包膜蛋白酶的作用外，还直接参与能量转导过程。TonB和ExbB在细胞质膜两侧的拓扑分布将这些蛋白质之间的相互作用位点主要限制在它们的跨膜结构域。在本研究中，TonB氨基末端跨膜锚中缬氨酸17的缺失导致TonB活性完全丧失，以及在体内检测不到与一个据信含有ExbB的59 kDa复合物的交联。ΔV17突变对TonB的输出没有影响。交联的丧失似乎反映了TonB/ExbB对的构象变化，而不是相互作用的丧失，因为ExbB对于TonB ΔV17的某些稳定作用仍然是必需的。分子建模表明，ΔV17突变导致TonB氨基末端膜锚预测的保守面发生显著变化。TonB ΔV17在裂解的原生质球中无法形成具有活性TonB特征的23 kDa蛋白酶K抗性形式。当没有ExbB时，野生型TonB也无法形成蛋白酶K抗性构型。综合这些结果表明，ΔV17突变中断了有效的TonB-ExbB相互作用。通过ExbB蛋白第一个预测跨膜结构域内或附近的第二个突变（A39E），恢复了与ExbB交联的明显能力以及有限的能量转导能力。与弱抑制一致，在存在ExbBA39E的情况下未观察到23 kDa蛋白酶K抗性形式的TonB ΔV17。ExbBA39E等位基因或ExbB的缺失均不影响TonB或TonB ΔV17的输出。与tonB ΔV17突变不同，exbBA39E突变并没有显著改变建模的ExbB跨膜结构域结构。此外，抑制子ExbBA39E与野生型TonB正常发挥作用，表明该抑制子不是等位基因特异性的。与预期相反，TonB ΔV17、ExbBA39E对导致TonB的半衰期大大缩短（约10分钟）。这些结果与蛋白酶敏感性研究一起表明，ExbB通过调节TonB的构象发挥作用。
