Stolle Patrick, Hou Bo, Brüser Thomas
From the Institute of Microbiology, Leibniz Universität Hannover, Herrenhäuser Strasse 2, 30419 Hannover, Germany.
From the Institute of Microbiology, Leibniz Universität Hannover, Herrenhäuser Strasse 2, 30419 Hannover, Germany
J Biol Chem. 2016 Jun 24;291(26):13520-8. doi: 10.1074/jbc.M116.729103. Epub 2016 Apr 22.
In Escherichia coli, cytoplasmic copper ions are toxic to cells even at the lowest concentrations. As a defense strategy, the cuprous oxidase CueO is secreted into the periplasm to oxidize the more membrane-permeable and toxic Cu(I) before it can enter the cytoplasm. CueO itself is a multicopper oxidase that requires copper for activity. Because it is transported by the twin-arginine translocation (Tat) pathway, which transports folded proteins, a requirement for cofactor assembly before translocation has been discussed. Here we show that CueO is transported as an apo-protein. Periplasmic CueO was readily activated by the addition of copper ions in vitro or under copper stress conditions in vivo Cytoplasmic CueO did not contain copper, even under copper stress conditions. In vitro Tat transport proved that the cofactor assembly was not required for functional Tat transport of CueO. Due to the post-translocational activation of CueO, this enzyme contributes to copper resistance not only by its cuprous oxidase activity but also by chelation of copper ions before they can enter the cytoplasm. Apo-CueO was indistinguishable from holo-CueO in terms of secondary structural elements. Importantly, the binding of copper to apo-CueO greatly stabilized the protein, indicating a transformation from an open or flexible domain arrangement with accessible copper sites to a closed structure with deeply buried copper ions. CueO is thus the first example for a natural Tat substrate of such incomplete folding state. The Tat system may need to transport flexibly folded proteins in any case when cofactor assembly or quaternary structure formation occurs after transport.
在大肠杆菌中，细胞质中的铜离子即使在最低浓度下对细胞也是有毒的。作为一种防御策略，亚铜氧化酶CueO被分泌到周质中，以便在毒性更强且更易透过膜的Cu(I)进入细胞质之前将其氧化。CueO本身是一种多铜氧化酶，其活性需要铜。由于它是通过双精氨酸转运（Tat）途径运输的，该途径运输折叠后的蛋白质，因此有人讨论过在转运之前是否需要辅因子组装。在这里，我们表明CueO是以脱辅基蛋白的形式被运输的。体外添加铜离子或体内处于铜胁迫条件下时，周质中的CueO很容易被激活。即使在铜胁迫条件下，细胞质中的CueO也不含铜。体外Tat转运证明，CueO的功能性Tat转运不需要辅因子组装。由于CueO的转运后激活，这种酶不仅通过其亚铜氧化酶活性，还通过在铜离子进入细胞质之前对其进行螯合，从而有助于提高铜抗性。就二级结构元件而言，脱辅基CueO与全酶CueO没有区别。重要的是，铜与脱辅基CueO的结合极大地稳定了该蛋白，表明其从具有可及铜位点的开放或灵活结构域排列转变为铜离子深埋的封闭结构。因此，CueO是这种不完全折叠状态的天然Tat底物的第一个例子。在任何情况下，当辅因子组装或四级结构形成在转运后发生时，Tat系统可能都需要运输灵活折叠的蛋白质。
