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J. Biol. Chem., Vol. 281, Issue 46, 35413-35424, November 17, 2006

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Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD^*

David M. Carter¹, Isabelle R. Miousse², Jean-Nicolas Gagnon³, Éric Martinez⁴, Abigail Clements⁵, Jongchan Lee, Mark A. Hancock, Hubert Gagnon, Peter D. Pawelek⁶, and James W. Coulton⁷

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

For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent K_D of 300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with K_D values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.

Received for publication, August 9, 2006

^* This work was supported in part by Operating Grant MOP-62774 (to J. W. C.) from the 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.

¹ Recipient of the McGill Graduate Studies Fellowship and an F. C. Harrison Fellowship.

² Recipient of a Natural Sciences and Engineering Research Council (Canada) Undergraduate Student Research Award.

³ Recipient of a postgraduate scholarship.

⁴ Trainee from theÉ_cole Supérieure de Biotechnologie, Strasbourg, France. Present address: Dept. of Life Science, Imperial College, London, UK.

⁵ Recipient of a traveling award for research training from the National Health and Medical Research Council (Australia). Present address: Dept. of Microbiology and Immunology, The University of Melbourne, Parkville, Australia.

⁶ Present address: Dept. of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H4B 1R6, Canada.

⁷ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, 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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