Abstract
The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). We genetically introduced pairs of Cys residues in different regions of the FepA tertiary structure, with the potential to form disulfide bonds. These included Cys pairs on adjacent β-strands of the N-domain (intra-N) and Cys pairs that bridged the external surface of the N-domain to the interior of the C-terminal transmembrane β-barrel (inter-N-C). We characterized FeEnt uptake by these mutants with siderophore nutrition tests, [59Fe]Ent binding and uptake experiments, and fluorescence decoy sensor assays. The three methods consistently showed that the intra-N disulfide bonds, which restrict conformational motion within the N-domain, prevented FeEnt uptake, whereas most inter-N-C disulfide bonds did not prevent FeEnt uptake. These outcomes indicate that conformational rearrangements must occur in the N terminus of FepA during FeEnt transport. They also argue against disengagement of the N-domain out of the channel as a rigid body and suggest instead that it remains within the transmembrane pore as FeEnt enters the periplasm.
Highlights
The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain)
Some Cys pairs (33–120, 42–162, 56 –73, and 149 –180; Fig. 2A) showed no evidence of disulfide bond formation in the gels or immunoblots; i.e. the electrophoretic mobility of those double-Cys derivatives exactly matched that of WT FepA, despite the fact that other experiments showed that they were disulfide-bonded
The phenotypes of the Cyspair mutants under investigation led to the same conclusion: intra-N disulfide bonds generally prevented FeEnt transport, whereas inter-N–C disulfide bonds generally did not prevent FeEnt transport (Fig. 1)
Summary
The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). Previous studies concluded that the 150-residue N-terminal globular domain (N-domain) likely exits the C-domain channel into the periplasm during ligand uptake [29, 36, 37] These results did not address how the N-domain moves or changes in structure to promote passage of metal complexes or other molecules [38, 39] through the transmembrane pore. The experiments found that intra-N disulfide bonds prevented FeEnt transport, but the majority of interN–C disulfides did not impair iron uptake These findings indicate that the N terminus of FepA must rearrange during FeEnt uptake, does not exit the channel as a rigid body, and may instead remain within the pore as it allows or promotes passage of FeEnt into the periplasm
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