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M506708200v1
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Papers In Press, published online ahead of print June 30, 2005
J. Biol. Chem, 10.1074/jbc.M506708200
Submitted on June 20, 2005
Accepted on June 30, 2005

Siderophore transport through Escherichia coli outer membrane receptor FhuA with disulfide-tethered cork and barrel domains

H. Anne Eisenhauer, Sofia Shames, Peter D. Pawelek, and James W. Coulton

Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4

Corresponding Author: james.coulton{at}mcgill.ca

Hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded ß-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double-cysteine mutants FhuACys109,356 and FhuACys112,383 would form disulfide bonds, thereby tethering the cork and barrel domains. Double-cysteine FhuA mutants were denatured under non-reducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double-cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of cork to barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway did not abrogate ferricrocin uptake. We propose that during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.


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