Larsen R A, Thomas M G, Postle K
Department of Microbiology, Washington State University, Pullman 99164-4233, USA.
Mol Microbiol. 1999 Mar;31(6):1809-24. doi: 10.1046/j.1365-2958.1999.01317.x.
TonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB-delta V17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-delta V17, for which both the conserved motif and the register of the predicted transmembrane domain alpha-helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant-suppressor pairs. The phenotype of TonB-delta V17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-delta V17. Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.
托蛋白（TonB）将细胞质膜质子动力（pmf）与跨外膜的主动运输相偶联，这一过程可能是通过一系列构象变化来实现的。先前对托蛋白跨膜结构域突变体（TonB-ΔV17）及其表型抑制子（ExbB-A39E）的研究表明，托蛋白对构象敏感。在此，分离出了保守的托蛋白跨膜结构域SHLS基序的两个新突变，即TonB-S16L和-H20Y，以及两个新的抑制子，即ExbB-V35E和-V36D。每个抑制子ExbB都至少部分恢复了托蛋白突变体的功能，不过，保守基序和预测的跨膜结构域α-螺旋的排列都受到影响的TonB-ΔV17是最难恢复功能的。如先前所示，只要存在ExbB和功能性的托蛋白跨膜结构域，托蛋白就能经历至少一种构象变化。在此，我们表明这种构象变化反映了托蛋白对细胞质膜质子梯度作出反应的能力，并且与突变体-抑制子对所达到的托蛋白活性水平成比例发生。TonB-ΔV17的表型比-S16L和-H20Y突变更为复杂，因为除了无法有效获得能量外，它还存在条件性不稳定性。只有在被ExbB突变体抑制后，这种第二个缺陷才明显，ExbB突变体使跨膜结构域突变体能够获得能量，并表现为TonB-ΔV17的快速周转。重要的是，这种降解依赖于托蛋白依赖性配体的存在，这表明在能量转导事件后托蛋白的构象也会发生变化。总之，这些观察结果支持了一种能量转导的动态模型，其中托蛋白通过一组势能不同的构象循环，向更高能量状态的转变由pmf驱动，而向更低能量状态的转变伴随着储存的势能释放到外膜受体。
