Abstract 1502 Investigations of Vibrio cholerae ferrous iron transport protein B (FeoB) nucleotide specificity

Ferrous iron (Fe2+) is an essential cofactor involved in several critical metabolic and biochemical processes. The presence of Fe2+ is also key to the virulence of numerous pathogenic bacteria such as Vibrio cholerae, the causative agent of the disease cholera. Pathogenic bacteria such as V. cholerae acquire Fe2+ from the host environment via the ferrous iron transport (Feo) system, the most widely conserved Fe2+ acquisition system across the prokaryotic domain. The most conserved component of the Feo system is FeoB, a polytopic transmembrane protein that bears a soluble N-terminal G-protein-like domain termed NFeoB, which facilitates the translocation of Fe2+ across the lipid bilayer. Although numerous crystal structures of NFeoB exist from several bacterial homologs, the structure of the V. cholerae NFeoB domain was previously unknown. Intriguingly, V. cholerae NFeoB exhibits nucleotide promiscuity, hydrolyzing both GTP and ATP, but the structural basis of this phenomenon remains unclear. We have recently determined the X-ray crystal structures of apo and GDP-bound V. cholerae NFeoB. Using this structural information, several variants of V. cholerae NFeoB were then generated based on the hypothesis that these amino acids are critical for nucleotide binding. In this work, we report the overproduction, purification, and oligomeric analyses of each V. cholerae NFeoB variant. We then analyzed the nucleotide binding capacity of each V. cholerae NFeoB variant using isothermal titration calorimetry (ITC). Finally, we tested each variant for its ability to crystallize and its X-ray diffraction capabilities. Coupled with our recent apo and GDP-bound structures of the wild-type domain, these results begin to elucidate the nucleotide promiscuity of the V. cholerae FeoB protein, which could be targeted in the future for therapeutic developments to treat cholera. NSF CAREER1844624.