Conformational rearrangements in the N-domain of Escherichia coli FepA during ferric enterobactin transport

• Published in: The Journal of biological chemistry 2020-04, Vol.295 (15), p.4974-4984
• Main Authors: Majumdar, Aritri, Trinh, Vy, Moore, Kyle J., Smallwood, Chuck R., Kumar, Ashish, Yang, Taihao, Scott, Daniel C., Long, Noah J., Newton, Salete M., Klebba, Phillip E.
• Format: Article
• Language: English
• Subjects: Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Biological Transport; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; disulfide; Enterobactin - metabolism; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; FepA; fluorescent sensor; iron; Membrane Biology; membrane transport; Models, Molecular; Mutagenesis, Site-Directed; Mutation; outer membrane; Protein Binding; Protein Conformation; Protein Domains; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; siderophore; site-directed mutagenesis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.011850

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.