Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System
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.
- Research Organization:
- SLAC National Accelerator Lab., Menlo Park, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC02-76SF00515
- OSTI ID:
- 953963
- Report Number(s):
- SLAC-REPRINT-2009-429; JBCHA3; TRN: US201004%%700
- Journal Information:
- J. Biol. Chem 282:35695,2007, Vol. 282, Issue 49; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
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
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CHEM