Transport Biochemistry of FepA

Abstract

This chapter provides an overview of structural features of metal transporters as a basis for mechanistic discussions that follow. The crystals of FepA, FhuA, FecA, and BtuB all showed the hollow, 22-strand antiparallel β-barrel of the C-domain, which spans the outer membrane (OM) and contains large extracellular loops that function in ligand binding, and the structurally distinct globular N-domain, which folds into the barrel interior, blocking access to the periplasm. The FepA crystal structure did not illustrate the disposition of bound ligands, but it is known from site-directed mutagenesis and biochemistry that basic and aromatic amino acids in the surface loops adsorb ligands on the basis of ionic and hydrophobic bonds and ring stacking. FepA interconverts between two distinct structural forms that were seen by electron spin resonance (ESR) spectroscopy. ESR spectroscopy also observed conformational changes in FepA during ligand uptake, but its low sensitivity made the technique difficult to employ in vivo. Ferric enterobactin (FeEnt) binds to FepA with a subnanomolar Kd that translates into a dissociation half-life of over 1 min. This calculation conflicts with the experimentally observed 20-s turnover number of FepA and argues for protein conformational change as an essential part of the transport mechanism that (i) undermines the affinity of the siderophore-receptor binding interaction, (ii) originates or unveils a channel to the periplasm, and (iii) facilitates internalization of the metal complex from the surface loops through the pore.