Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system

Ireena Bagai, Wenbo Liu, Christopher Rensing, Ninian Blackburn, Megan M. McEvoy

Research output: Contribution to journalArticle

65 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)35695-35702
Number of pages8
JournalJournal of Biological Chemistry
Volume282
Issue number49
DOIs
StatePublished - Dec 7 2007

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Metals
Substrates
Methionine
Copper
X-Ray Absorption Spectroscopy
Periplasmic Proteins
Calorimetry
X ray absorption spectroscopy
Isoleucine
Chromatography
Gram-Negative Bacteria
Titration
Silver
Sulfur
Gel Chromatography
Sequence Analysis
Bacteria
Gels
Ions
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system. / Bagai, Ireena; Liu, Wenbo; Rensing, Christopher; Blackburn, Ninian; McEvoy, Megan M.

In: Journal of Biological Chemistry, Vol. 282, No. 49, 07.12.2007, p. 35695-35702.

Research output: Contribution to journalArticle

Bagai, Ireena ; Liu, Wenbo ; Rensing, Christopher ; Blackburn, Ninian ; McEvoy, Megan M. / Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 49. pp. 35695-35702.
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