The TonB-dependent receptors (TBDRs) FepA and FhuA transport the siderophores ferric enterobactin (FeEnt) and ferrichrome (Fc), respectively, through the Gram-negative bacterial outer membrane. Their uptake mechanism involves conformational change in an ~150 residue N-terminal luminal domain (NTLD), located within their C-terminal β-barrel (CTβB) channels. We identified four internal sites (1–4) in TBDR that form a conserved network of ion pairs encircling the NTLD-CTβB interface. We tested the mechanistic importance of these electrostatic interactions by engineering systematic Ala substitutions in FepA and FhuA for the acidic or basic side chains that comprise them. Siderophore nutrition assays, colicin susceptibility tests and fluorescence spectroscopic uptake measurements of the mutants showed the importance of site-2, that adheres the base of NL1/Nβ3 and Nβ5 of the NTLD to β14 and β17 on the interior of the CTβB. Disruption of electrostatic bonds at site-2 reduced or eliminated ferric siderophore uptake and severely curtailed colicin susceptibility. Despite these reductions in ligand transport, fluorescent spectroscopic binding measurements showed that the site-2 mutations did not alter the affinity of FepA for FeEnt, nor FhuA for Fc. Elimination of ionic interactions at the three other locations in FepA (sites-1, -3, -4) did not reduce FeEnt uptake. Lastly, in FepA the disruption of ionic bonding at site-2 in FepA rendered it more susceptible to proteolysis, in part by OmpT, suggesting that ablation of ionic interactions in site-2 destabilized the NTLD within the CTβB. Overall, the experiments demonstrated that the ion pairs at site-2 in FepA and FhuA, that are evolutionarily conserved in the TBDR superfamily, are essential to the movement of ferric siderophores through the CTβB into the periplasm.
Newton et al. (Fri,) studied this question.