From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle
From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle
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DOI：
10.1128/jb.00484-15
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发表时间：
2015-11-01
影响因子：
3.2
通讯作者：
Postle, Kathleen
中科院分区：
文献类型：
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作者：
Gresock, Michael G.;Kastead, Kyle A.;Postle, Kathleen
TonB系统利用细胞质膜（CM）的质子梯度通过革兰氏阴性菌的外膜（OM）转运蛋白主动转运大量、稀缺且重要的营养物质。在大肠杆菌中，CM蛋白ExbB和ExbD利用质子动力能并将其转移至CM蛋白TonB，其跨越周质空间并循环结合OM转运蛋白。TonB有两个活动域：具有残基H20的氨基末端跨膜结构域和周质羧基末端，通过其结合OM转运蛋白。TonB通过残基H20处除H20N之外的所有取代而失活。在这里，我们表明，虽然通过其氨基末端结构域作为同源二聚体捕获TonB保留全部活性，通过其羧基末端捕获TonB通过防止与OM转运蛋白相互作用所需的构象变化使其失活。令人惊讶的是，无活性的TonB H20A对通过氨基末端的同源二聚化几乎没有影响，而是在能量转导循环重新开始之前降低了TonB羧基末端同源二聚体的形成。该结果表明TonB羧基末端最终作为单体与OM转运蛋白相互作用。我们的研究结果还表明存在不与TonB接触的ExbD同源二聚体的单独等摩尔池。提出了一种模型，其中TonB同二聚体与ExbD同二聚体的相互作用启动能量转导循环，并且最终，ExbD羧基末端调节单体TonB羧基末端与OM转运蛋白的相互作用。在TonB将其与ExbD的相互作用交换为与转运蛋白的相互作用后，ExbD同源二聚体经历了重新激活它们所需的单独循环。重要的是，跨细胞质膜主动转运的典型机制利用离子梯度或ATP水解来获得能量。革兰氏阴性细菌的外膜缺乏这些资源。TonB系统体现了一种新的跨外膜主动运输的方式，用于太大、太稀缺或对扩散限制运输太重要的营养物质。跨细胞质膜的质子梯度被多蛋白复合物转化为机械能，该机械能驱动跨外膜的高亲和力主动运输。该系统也是令人感兴趣的，因为其在病原菌中的用途之一是与宿主竞争必需元素铁。了解TonB系统的机制将允许设计针对铁获取的抗生素。
The TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). In Escherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them.IMPORTANCECanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.