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J. Biol. Chem., Vol. 282, Issue 49, 35695-35702, December 7, 2007

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Substrate-linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System^*

Ireena Bagai, Wenbo Liu, Christopher Rensing, Ninian J. Blackburn^¶, and Megan M. McEvoy¹

From the Department of Biochemistry and Molecular Biophysics and Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, Arizona 85721 and the ^¶Department of Environmental and Biomolecular Systems, Oregon Graduate Institute School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon 97006-8921

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.

Received for publication, May 14, 2007 , and in revised form, September 20, 2007.

^* This work was supported by National Institutes of Health Grants GM54803 (to N. J. B.) and GM079192 (to M. M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: 1041 E. Lowell St., Dept. of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721. Fax: 520-621-1697; E-mail: mcevoy{at}email.arizona.edu.

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