Abstract
The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-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.
Highlights
Iron is required by most living cells because of its diverse roles in numerous metabolic processes, including glycolysis, energy generation by electron transport, and DNA synthesis [1]
The hydroxamate siderophore receptor FhuA is a TonBdependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded -barrel occluded by an N-terminal globular cork domain
Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains
Summary
Iron is required by most living cells because of its diverse roles in numerous metabolic processes, including glycolysis, energy generation by electron transport, and DNA synthesis [1]. Conserved residues along the inner barrel wall of the putative siderophore transport pathway may facilitate the diffusion of ferrichrome through FhuA by a series of low affinity binding interactions. Such a mechanism was shown for diffusion of maltose and maltodextrins along the greasy slide of LamB porin [10]. It has been postulated that displacement of the siderophore from its initial binding site drives an inward motion of the extracellular loops, resulting in removal of the cork from the barrel lumen [4] In this model, the siderophore could pass through a transiently formed pathway by diffusion. It has even been suggested that the cork domain may physically import bound ligands through the barrel during its own translocation into the periplasm [6, 16]
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