The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain

Abstract

Most pathogenic bacteria require ferrous iron (Fe 2+ ) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly-conserved prokaryotic transporter of Fe 2+ , but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein and FeoB, a membrane protein that translocates Fe 2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that select FeoC proteins bind O 2 -sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O 2 to regulate Fe 2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera ( Vibrio cholerae ) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of Vc FeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of Vc FeoC reveals a truncated winged β-sheet in which the cluster-binding domain is notably absent. To test the interactions of Vc FeoC with Vc FeoB, we used NMR to demonstrate that these proteins interact in a 1:1 stoichiometry with a K d of ca . 25 μM. Finally, using homology modeling, we predicted the structure of Vc NFeoB and used docking to identify an interaction site with Vc FeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of Vc FeoC and contribute to a better understanding of its role vis-à-vis FeoB.