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Title: Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System

Abstract

Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.

Authors:
; ; ; ;
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
953963
Report Number(s):
SLAC-REPRINT-2009-429
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US201004%%700
DOE Contract Number:  
AC02-76SF00515
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem 282:35695,2007
Additional Journal Information:
Journal Volume: 282; Journal Issue: 49; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; ABSORPTION SPECTROSCOPY; AFFINITY; BACTERIA; CALORIMETRY; CHROMATOGRAPHY; CONFORMATIONAL CHANGES; FILTRATION; FUNCTIONALS; GELS; IN VITRO; IONS; MEMBRANES; METALS; METHIONINE; MOLECULES; MUTATIONS; PLATES; PROTEINS; RESIDUES; STRUCTURAL CHEMICAL ANALYSIS; SUBSTRATES; TITRATION; Other,BIO, CHEM

Citation Formats

Bagai, I, Liu, W, Rensing, C, Blackburn, N J, and McEvoy, M M. Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System. United States: N. p., 2009. Web.
Bagai, I, Liu, W, Rensing, C, Blackburn, N J, & McEvoy, M M. Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System. United States.
Bagai, I, Liu, W, Rensing, C, Blackburn, N J, and McEvoy, M M. 2009. "Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System". United States.
@article{osti_953963,
title = {Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System},
author = {Bagai, I and Liu, W and Rensing, C and Blackburn, N J and McEvoy, M M},
abstractNote = {Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.},
doi = {},
url = {https://www.osti.gov/biblio/953963}, journal = {J. Biol. Chem 282:35695,2007},
issn = {0021-9258},
number = 49,
volume = 282,
place = {United States},
year = {Tue Jun 02 00:00:00 EDT 2009},
month = {Tue Jun 02 00:00:00 EDT 2009}
}