Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

Abstract

Gram-negative bacteria, such as Escherichia coli, use tripartite efflux complexes in the resistance-nodulation-division family to expel diverse toxic compounds from the cell. The CusCBA system is responsible for extruding biocidal Cu(I) and Ag(I) ions from the cell. The X-ray crystal structure of CusA, the inner membrane transporter, has now been determined in the absence and presence of bound Cu(I) or Ag(I). The structures suggest that the metal binding site, formed by a three-methionine cluster, is located within the cleft region of the periplasmic domain. The authors propose a potential pathway for ion export in which CusA is capable of picking up the metal ion from the cytosol, with this transporter utilizing the methionine pairs and clusters to bind and export metal ions. Gram-negative bacteria, such as Escherichia coli, use tripartite efflux complexes in the resistance-nodulation-cell division family to expel toxic compounds from the cell. The CusCBA system is responsible for removing biocidal Cu(I) and Ag(I) ions. Here, the X-ray crystal structure is reported of CusA in the absence and presence of bound Cu(I) or Ag(I). The structures reveal that the metal-binding sites are located within the cleft region of the periplasmic domain. A potential pathway for ion export is proposed. 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 cell1,2. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions3,4. 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.