Molecular, Cellular and Developmental Biology Interdepartmental Graduate Program, Iowa State University, Iowa 50011, USA.
Nature. 2010 Sep 23;467(7314):484-8. doi: 10.1038/nature09395.
Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs-three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.
革兰氏阴性菌，如大肠杆菌，经常使用三部分流出复合物在耐药性结节分裂（RND）家族中，将各种有毒化合物从细胞中排出。流出系统 CusCBA 负责排出杀菌的 Cu（I）和 Ag（I）离子。以前没有关于 RND 流出泵的重金属流出（HME）亚家族的结构信息。在这里，我们描述了在没有结合 Cu（I）或 Ag（I）的情况下，内膜转运蛋白 CusA 的晶体结构。这些 CusA 结构提供了有关 RND 流出泵的 HME 亚家族的新结构信息。这些结构表明，金属结合位点由三个蛋氨酸簇形成，位于周质域的裂缝区域内。该裂缝在 apo-CusA 形式中关闭，但在 CusA-Cu（I）和 CusA-Ag（I）结构中打开，这直接表明了离子出口的合理途径。Cu（I）和 Ag（I）的结合引发了周质和跨膜结构域的显著构象变化。晶体结构表明，CusA 除了三个蛋氨酸金属结合位点外，还有四个蛋氨酸对-三个位于跨膜区域，一个位于周质域。遗传分析和转运试验表明，CusA 能够从细胞质中主动摄取金属离子，使用这些蛋氨酸对或簇来结合和输出金属离子。这些结构表明，在这些位点之间存在逐步穿梭机制。
