Effect of loop deletions on the binding and transport of ferric enterobactin by FepA

The siderophore ferric enterobactin enters Escherichia coli through the out er membrane (OM) porin FepA, which contains an aqueous transmembrane channe l that is normally occluded by other parts of the protein. After binding th e siderophore at a site within the surface loops, FepA undergoes conformati onal changes that promote ligand internalization. We assessed the participa tion of different loops in ligand recognition and uptake by creating and an alysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM p rotein, The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (K-d) and uptake (K-m): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficie ncy. We classified the mutations in three groups on the basis of their slig ht, strong or complete inhibition of the rate of ferric enterobactin transp ort across the OM, Finally, characterization of the FepA mutants revealed t hat prior experiments underestimated the affinity of FepA for ferric entero bactin: the interaction between the protein and the ferric siderophore is s o avid (K-d < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality, That is, like other porins, many of the loops of FepA ate superficially dispensa ble: ferric enterobactin transport occurred without them, at levels that al lowed bacterial growth.