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

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. In this paper, 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. Finally, our results suggest that metal transfermore » 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.« less
Authors:
 [1] ;  [2] ;  [2] ;  [1]
  1. Oregon Health and Science Univ., Portland, OR (United States)
  2. Univ. of Arizona, Tucson, AZ (United States)
Publication Date:
Grant/Contract Number:
DGE-0925180; R01 GM054803; R01 079192
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 111; Journal Issue: 43; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Oregon Health and Science Univ., Portland, OR (United States); Univ. of Arizona, Tucson, AZ (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Inst. of Health (NIH) (United States); National Science Foundation (NSF)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; copper; periplasmic efflux; X-ray absorption spectroscopy; metal ion transport; host–pathogen interaction
OSTI Identifier:
1349641

Chacon, Kelly N., Mealman, Tiffany D., McEvoy, Megan M., and Blackburn, Ninian J.. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins. United States: N. p., Web. doi:10.1073/pnas.1411475111.
Chacon, Kelly N., Mealman, Tiffany D., McEvoy, Megan M., & Blackburn, Ninian J.. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins. United States. doi:10.1073/pnas.1411475111.
Chacon, Kelly N., Mealman, Tiffany D., McEvoy, Megan M., and Blackburn, Ninian J.. 2014. "Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins". United States. doi:10.1073/pnas.1411475111. https://www.osti.gov/servlets/purl/1349641.
@article{osti_1349641,
title = {Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins},
author = {Chacon, Kelly N. and Mealman, Tiffany D. and McEvoy, Megan M. and Blackburn, Ninian J.},
abstractNote = {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. In this paper, 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. Finally, 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.},
doi = {10.1073/pnas.1411475111},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 43,
volume = 111,
place = {United States},
year = {2014},
month = {10}
}