Concerted Loop Motion Triggers Induced Fit of FepA to Ferric Enterobactin (LB231)

Abstract

By spectroscopic analyses of extrinsic fluorophore_labeled site‐directed Cys residues in 7 surface loops of Escherichia coli FepA, we characterized the binding and transport of ferric enterobactin (FeEnt). Changes in fluorescence emissions reflected conformational motion of loops that altered the environment of the fluorophore, and we observed these dynamics as quenching phenomena during FeEnt binding and transport in living cells or outer membrane vesicles. Cys residues in each of the 7 surface loops (L2, L3, L4, L5, L7 L8, and L11) behaved individually and characteristically with regard to both fluorophore maleimide reactivity and conformational motion. During FeEnt binding the loops closed around FeEnt in approximately one second, in the order L11 > L3 > L2 > L7 > L8 > L5 > L4. This sequence created a hierarchy that correlated with the structure of FepA: L11, L3 and L2 sit opposite L7, L8, L5 and L4 across the outer vestibule, suggesting that the loops bind the metal complex similar to two hands clasping around an object. Although L3 followed a distinctly biphasic exponential decay during FeEnt binding, fluorescence from the other loops followed simpler, single exponential decay processes. Fluorescence measurements of FeEnt transport, by either microscopic or spectroscopic methodologies, demonstrated that ligand uptake occurs uniformly throughout the cell envelope, and susceptibility of FeEnt uptake to the proton ionophore m‐chlorophenyl hydrazone (CCCP) at concentrations as low as 5 uM. The latter result recapitulates the sensitivity of inner membrane major facilitator transporters to CCCP (Kaback, 1974), providing further evidence of the electrochemical gradient as a driving force for TonB‐dependent metal transport.Grant Funding Source: Supported by National Institutes of Health Grant GM53836 and National Science Foundation Grant MCB09522999