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J. Biol. Chem., Vol. 280, Issue 34, 30574-30580, August 26, 2005

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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

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

The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded -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 L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing 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 the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.

Received for publication, June 20, 2005

^* This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research (to J. W. C.). The Canada Foundation for Innovation provided infrastructure for fluorescence imaging and densitometry to the Montreal Integrated Genomics Group for Research on Infectious Pathogens. The Sheldon Biotechnology Centre at McGill University is supported by a multi-user maintenance grant from Canadian Institutes of Health Research. 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.

To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Rm. 403, McGill University, 3775 University St., Montreal, Quebec H3A 2B4, Canada. Tel.: 514-398-3929; Fax: 514-398-7052; E-mail: james.coulton{at}mcgill.ca.

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