Abstract 2387 Biophysical characterization of the Vibrio cholerae FeoABC complex

The acquisition of ferrous iron (Fe2+) is important for the establishment of infection via several pathogenic prokaryotes. The ferrous iron transport (Feo) system is the most widely conserved system employed for Fe2+ uptake, and several bacterial species become avirulent in the absence of components of this multi-protein system. The Feo system is canonically tripartite and consists of: FeoA, FeoB, and FeoC. FeoA and FeoC are both small (ca. 8 kDa), cytoplasmic proteins, while FeoB is a large (ca. 85 kDa) polytopic transmembrane protein with a soluble, N-terminal domain (NFeoB) that hydrolyzes NTPs and is thought to control Fe2+ translocation. Based on work that examined the Feo system within Vibrio cholerae, FeoA and FeoC are both known to interact with NFeoB in vivo. We have recently determined the structures of VcFeoC and VcNFeoB, and we have shown that VcFeoC interacts weakly with intact VcFeoB in vitro. However, the atomic-level details regarding how FeoA, NFeoB, and FeoC interact to form a complex are unknown. To tackle this important problem, I have first developed purification protocols to deliver large amounts each soluble component of the V. cholerae Feo system (VcFeoA, VcNFeoB, and VcFeoC) that are both highly pure and highly homogeneous. Using size-exclusion chromatography, I have shown that VcFeoA and VcNFeoB interact as do VcFeoC and VcNFeoB, each in the absence of exogenous nucleotide. Based on these gel filtration data, a simple 1:1 stoichiometry for both complexes that are not in dynamic equilibrium is suggested. Additional biophysical and structural analyses such as isothermal titration calorimetry (ITC) and X-ray crystallography are currently underway to determine the strength of these interactions as well as the molecular details of how these interactions occur. The results from this work will provide the first atomic-level information demonstrating how FeoA and FeoC interact with NFeoB to regulate Feo function, which could be leveraged in the future in the design of novel therapeutics to treat diseases caused by Vibrio cholerae such as cholera.