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

Abstract

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:

Chacon, Kelly N. ^[1]; Mealman, Tiffany D. ^[2]; McEvoy, Megan M. ^[2]; Blackburn, Ninian J. ^[1]

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+ Show Author Affiliations

1. Oregon Health and Science Univ., Portland, OR (United States)
2. Univ. of Arizona, Tucson, AZ (United States)

Publication Date:

Mon Oct 13 00:00:00 EDT 2014

Research Org.:

Oregon Health & 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); National Inst. of Health (NIH) (United States); National Science Foundation (NSF)

OSTI Identifier:

1349641

Grant/Contract Number:  

DGE-0925180; R01 GM054803; R01 079192

Resource 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)

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