Recognition of Ferric Catecholates by FepA

Abstract

ABSTRACT Escherichia coli FepA transports certain catecholate ferric siderophores, but not others, nor any noncatecholate compounds. Direct binding and competition experiments demonstrated that this selectivity originates during the adsorption stage. The synthetic tricatecholate Fe-TRENCAM bound to FepA with 50- to 100-fold-lower affinity than Fe-enterobactin (FeEnt), despite an identical metal center, and Fe-corynebactin only bound at much higher concentrations. Neither Fe-agrobactin nor ferrichrome bound at all, even at concentrations 10 6 -fold above the K d . Thus, FepA only adsorbs catecholate iron complexes, and it selects FeEnt among even its close homologs. We used alanine scanning mutagenesis to study the contributions of surface aromatic residues to FeEnt recognition. Although not apparent from crystallography, aromatic residues in L3, L5, L7, L8, and L10 affected FepA's interaction with FeEnt. Among 10 substitutions that eliminated aromatic residues, K d increased as much as 20-fold (Y481A and Y638A) and K m increased as much as 400-fold (Y478), showing the importance of aromaticity around the pore entrance. Although many mutations equally reduced binding and transport, others caused greater deficiencies in the latter. Y638A and Y478A increased K m 10- and 200-fold more, respectively, than K d . N-domain loop deletions created the same phenotype: Δ60-67 (in NL1) and Δ98-105 (in NL2) increased K d 10- to 20-fold but raised K m 500- to 700-fold. W101A (in NL2) had little effect on K d but increased K m 1,000-fold. These data suggested that the primary role of the N terminus is in ligand uptake. Fluorescence and radioisotopic experiments showed biphasic release of FeEnt from FepA. In spectroscopic determinations, k off1 was 0.03/s and k off2 was 0.003/s. However, FepAY272AF329A did not manifest the rapid dissociation phase, corroborating the role of aromatic residues in the initial binding of FeEnt. Thus, the β-barrel loops contain the principal ligand recognition determinants, and the N-domain loops perform a role in ligand transport.