Disulphide trapping of an in vivo energy-dependent conformation of Escherichia coli TonB protein
Disulphide trapping of an in vivo energy-dependent conformation of Escherichia coli TonB protein
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DOI：
10.1111/j.1365-2958.2004.04384.x
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发表时间：
2005-01-01
影响因子：
3.6
通讯作者：
Postle, K
中科院分区：
文献类型：
--
作者：
Ghosh, J;Postle, K
在大肠杆菌中，TonB 系统可转换细胞质膜的质子动力 (pmf)，以支持各种跨外膜的转运事件。细胞质膜蛋白 ExbB 和 ExbD 似乎可以收获 pmf 并将其转导为 TonB。实验证据表明，TonB 穿梭至外膜，显然是将构象存储的势能传递给外膜转运蛋白。在最新的模型中，排出的 TonB 随后被回收到细胞质膜，由能量耦合蛋白 ExbB/D 重新提供能量。有人建议，活性 TonB 的羧基端 75 个氨基酸可以由相应片段的刚性、链交换、二聚晶体结构代表。相比之下，最近丙氨酸取代的遗传学研究表明，体内完整 TonB 的羧基末端是动态且灵活的。这里提出的生化研究证实并扩展了这些结果，证明一个单体亚基中芳香族残基上的单个半胱氨酸取代可以在体内与另一个单体亚基中的相同残基形成自发二聚体。两个通电的 TonB 似乎形成一个由 8-10 个芳香族氨基酸组成的簇，包括那些位于晶体结构两端的氨基酸。芳香簇需要 TonB 的氨基末端能量耦合结构域和 ExbB/D（以及串扰类似物 TolQ/R）来在体内形成。在细胞质膜相关的 TonB 中检测到大芳香族簇，但在外膜相关的 TonB 中未检测到。与这些观察结果一致，芳香族簇可以在能量转导循环的前半部分形成，然后将构象存储的势能释放到负载配体的外膜转运蛋白。出现的模型是，在通过 ExbB/D 和 TonB 跨膜结构域介导的 pmf 输入后，TonB 羧基末端可以形成一种亚稳定的高能构象，而该构象并不由羧基末端的晶体结构表示。
In Escherichia coli, the TonB system transduces the protonmotive force (pmf) of the cytoplasmic membrane to support a variety of transport events across the outer membrane. Cytoplasmic membrane proteins ExbB and ExbD appear to harvest pmf and transduce it to TonB. Experimental evidence suggests that TonB shuttles to the outer membrane, apparently to deliver conformationally stored potential energy to outer membrane transporters. In the most recent model, discharged TonB is then recycled to the cytoplasmic membrane to be re-energized by the energy coupling proteins, ExbB/D. It has been suggested that the carboxy-terminal 75 amino acids of active TonB could be represented by the rigid, strand-exchanged, dimeric crystal structure of the corresponding fragment. In contrast, recent genetic studies of alanine substitutions have suggested instead that in vivo the carboxy-terminus of intact TonB is dynamic and flexible. The biochemical studies presented here confirm and extend those results by demonstrating that individual cys substitution at aromatic residues in one monomeric subunit can form spontaneous dimers in vivo with the identical residue in the other monomeric subunit. Two energized TonBs appear to form a single cluster of 8-10 aromatic amino acids, including those found at opposite ends of the crystal structure. The aromatic cluster requires both the amino-terminal energy coupling domain of TonB, and ExbB/D (and cross-talk analogues TolQ/R) for in vivo formation. The large aromatic cluster is detected in cytoplasmic membrane-, but not outer membrane-associated TonB. Consistent with those observations, the aromatic cluster can form in the first half of the energy transduction cycle, before release of conformationally stored potential energy to ligand-loaded outer membrane transporters. The model that emerges is one in which, after input of pmf mediated through ExbB/D and the TonB transmembrane domain, the TonB carboxy-terminus can form a meta-stable high-energy conformation that is not represented by the crystal structure of the carboxy-terminus.