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


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.

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Figure 1: Structure of the apo CusA efflux pump.
Figure 2: Comparison of the apo and metal-bound structures of CusA.
Figure 3: Anomalous maps of the bound metal ions.
Figure 4: Proposed metal transport pathway of the CusA efflux pump.
Figure 5: Stopped-flow transport assay of reconstituted CusA with extravesicular Ag + ion.


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We thank M. D. Routh for critical reading of the manuscript. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source, supported by National Institutes of Health (NIH) award RR-15301 from the National Center for Research Resources. Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. This work was supported by NIH grants GM 074027 (to E.W.Y.), GM 086431 (to E.W.Y.), GM 081680 (to R.L.J.) and GM 072014 (to R.L.J.).

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F.L., C.-C.S. and E.W.Y. designed the research. F.L. and C.-C.S. performed experiments. M.T.Z. and S.E.B. performed simulations. C.-C.S., F.L., K.R.R. and E.W.Y. performed model building and refinement. F.L., C.-C.S., R.L.J. and E.W.Y. wrote the paper.

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Correspondence to Edward W. Yu.

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Long, F., Su, C., Zimmermann, M. et al. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport. Nature 467, 484–488 (2010).

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