Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

Kelly N. Chacón, Tiffany D. Mealman, Megan M. McEvoy, Ninian J. Blackburn

Research output: Contribution to journalArticlepeer-review

63 Scopus citations


Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host-pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. Here, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a "switch" role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.

Original languageEnglish (US)
Pages (from-to)15373-15378
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number43
StatePublished - Oct 28 2014


  • Copper
  • Host-pathogen interaction
  • Metal ion transport
  • Periplasmic efflux
  • X-ray absorption spectroscopy

ASJC Scopus subject areas

  • General


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