Biochemical and structural characterization of the fused Bacteroides fragilis NFeoAB domain reveals a role for FeoA

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system has been identified as the primary prokaryotic Fe2+ import machinery, and two proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remained enigmatic. In this work we explored the distribution of feoAB operons predicted to encode for a fusion of FeoA tethered to the soluble N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion might poise FeoA to interact with FeoB in order to affect function. To test this hypothesis, we cloned, expressed, purified, and biochemically characterized the soluble NFeoAB fusion protein from Bacteroides fragilis , a commensal organism implicated in drug-resistant peritoneal infections. Using X-ray crystallography, we determined to 1.50 Å resolution the structure of Bf FeoA, which adopts an SH3-like fold implicated in protein-protein interactions. In combination with structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB indeed interact in a nucleotide-dependent manner, and we have mapped the protein-protein interaction interface. Finally, using GTP hydrolysis assays, we demonstrate that Bf NFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis and is not potassium-stimulated, indicating that FeoA-NFeoB interactions may function to stabilize the GTP-bound form of FeoB. Our work thus reveals a role for FeoA function in the fused FeoAB systems and suggests a broader role for FeoA function amongst prokaryotes. ### Competing Interest Statement The authors have declared no competing interest. * ### Abbreviations GDP : guanosine diphosphate GMP-PNP : 5’guanylyl-imidodiphosphate GTP : guanosine triphosphate IMAC : immobilized metal affinity chromatography IPTG : isopropyl β-D-l-thiogalactopyranoside NFeoAB : soluble N-terminal GTP-binding domain of FeoB fused to FeoA NFeoB : soluble N-terminal GTP-binding domain of FeoB NMR : nuclear magnetic resonance PMSF : phenylmethylsulfonyl fluoride SAXS : small-angle X-ray scattering SDS-PAGE : sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC : size-exclusion chromatography SEC-SAXS : SEC-coupled small-angle X-ray scattering R.M.S.D. : root-mean-square deviation TEV : Tobacco Etch Virus Tris : tris(hydroxymethyl)aminomethane TCEP : tris(2-carboxyethyl)phosphine.