Recognition of ferric catecholates by FepA

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 Kd. 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, Kd increased as much as 20-fold (Y481A and Y638A) and Km 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 Km 10- and 200-fold more, respectively, than Kd. N-domain loop deletions created the same phenotype: Delta60-67 (in NL1) and Delta98-105 (in NL2) increased Kd 10- to 20-fold but raised Km 500- to 700-fold. W101A (in NL2) had little effect on Kd but increased Km 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 beta-barrel loops contain the principal ligand recognition determinants, and the N-domain loops perform a role in ligand transport.

FIG. 1.

(Top) Binding and transport. We compared the binding (open symbols) and transport (solid symbols) of ⁵⁹FeEnt (inverted triangles), ⁵⁹FeTrn (triangles), and FeCrn (diamonds) by E. coli strain KDF541/pITS23. (Bottom) Competition of ⁵⁹FeEnt binding to FepA by catecholate siderophores. We determined the abilities of ferrichrome (□), ferric agrobactin (○), FeTrn (▵), FeCrn (⋄), and FeEnt (▾) to inhibit the binding of ⁵⁹FeEnt to E. coli strain KDF541/pITS23 (fepA⁺). The data were analyzed and plotted by the IC50-4 Parameter Logistic of Grafit 5.09. The right panel shows the structures of Ent (A), Trn (B), Crn (C), agrobactin A (D), and apoferrichrome (E).

FIG. 2.

Site-directed FepA mutants. (A) Location. The model of FepA (14) depicts aromatic residues (yellow) that we changed to Ala, in space filling format viewed from the top (left). Hydrophobic residues (L, I, V, M, and A) are colored green. The locations of residues F329 and Y488 are not known (11), but the last solved residues in L4 (red) flank the approximate location of the former (yellow oval), and the last solved residues in L7 (light green) flank the approximate location of the latter residue (yellow oval). Basic residues are shown in CPK format. Residues of interest are also shown in space-filling format on a backbone representation viewed from the side (center), with the same color scheme. (Right) A backbone model, viewed from the top, shows the location of two site-directed deletions, Δ60-67 (red; in NL1) and Δ98-105 (orange; in NL2); NL2 contains residue W101 (yellow, in space-filling format). (B) Expression and localization in the OM. Cell lysates (top) or OM fractions (bottom) (58) from E. coli expressing wild-type FepA or its mutant derivatives were resolved by SDS-PAGE and either subjected to immunoblot analysis with anti-FepA MAb 45 and ¹²⁵I-protein A (top) or stained with Coomassie blue (bottom). The expression levels of the mutant proteins, and their localization to the OM were related to those of OmpF and OmpA and found to be comparable to that of wild-type FepA carried on the same plasmid. Lane 1 contains a sample from KDF541, and lanes 2 to 12 contain samples from KDF51 harboring pHSG575 carrying the fepA alleles fepA⁺, W101A, Y271A, Y472A, Y478A, Y481A, Y488A, Y495A, Y540A, Y553A, and Y638A. Lanes 13 to 15 contains samples from KDF541 harboring pUC19 carrying the fepA alleles fepA⁺, ΔNL1, and ΔNL2, respectively. (C) Siderophore nutrition tests. The site-directed mutations created several different effects in qualitative uptake assays. See the text for further explanation.

FIG. 3.

⁵⁹FeEnt binding and transport by Ala substitution mutants. We determined the concentration dependence of ⁵⁹FeEnt binding (open symbols) and transport (solid symbols) by E. coli strain KDF541 expressing FepA substitution mutant proteins carried on the low-copy plasmid pHSG575 at six concentrations near K_d and K_m, respectively, with each datum point collected in triplicate and averaged. The binding and transport data were analyzed, and curves were plotted by using the bound-versus-total and enzyme kinetics equations, respectively, of Grafit 5.09. The concentration of FeEnt is logarithmically plotted to demonstrate the decreases in affinity that some of the mutations caused. (Top) fepA⁺, circles; Y540A, triangles; and Y481A, squares. (Center) Y478A, inverted triangles; Y495A, diamonds. (Bottom) W101A, hexagons; Y638A, stars.

FIG. 4.

⁵⁹FeEnt binding and transport by N-loop deletion mutants. Methods were as described in Fig. 3. Open symbols indicate ⁵⁹FeEnt binding, and solid symbols indicate ⁵⁹FeEnt uptake by bacteria expressing fepA⁺ (circles), fepAΔ60-67 (squares), and fepAΔ98-105 (triangles) alleles. The binding and transport data were analyzed and curves were plotted by using the bound-versus-total and enzyme kinetics equations, respectively, of Grafit 5.09.

FIG. 5.

Measurements of FeEnt dissociation from FepA and FepAY272AF329A. Light gray symbols represent data derived from bacteria washed with LiCl; the mean of these data is plotted with an open symbol, and the fitted curve (double exponential decay) from these data is shown with a dashed line. Dark gray symbols represent data derived from bacteria washed with 50 μM FeEnt; the mean of these data is plotted with a black symbol, and the fitted curve (double exponential decay) from these data is shown with a solid line. (A) Release of ⁵⁹FeEnt from KDF541 (circles, fepA), BN1071 (squares, chromosomal fepA⁺), KDF541/pITS449 (triangles, plasmid fepA+). (B) Release of ⁵⁹FeEnt from KDF541/pfepAY272AF329A (diamonds). (C and D) KDF571 (tonB)/pfepAS271C was labeled with fluorescein maleimide, and the release of FeEnt in the presence of KDF571/pITS449 (blue) or KDF541/pITS449 (black) was measured spectrophotometrically. The red and green curves derive from nonlinear fits of the release data in the presence of KDF541/pITS449 by using equations for single or double exponential decays, respectively.

FIG. 6.

Model of FeEnt transport through FepA. (Top) Formal representation of the FepA transport process. Constants k₁ to k₄ are experimentally defined: fluors attached to L3 reflect both the first and second binding stages (k₁ = 0.02/s and k₃ = 0.005/s) (53), which are both reversible (k₂ = 0.03/s and k₄ = 0.003/s [the present study]). (Bottom) The FepA transport cycle is depicted as a series of conformational stages (17, 35, 60) that result in binding and internalization of FeEnt. The representations of FepA originated from its crystal structure, but they are postulated forms that were not crystallographically demonstrated. By analogy to FhuA and FecA, FeEnt binding may relocate the TonB-box region of FepA away from the β-barrel wall. Such movement may signal receptor occupancy to TonB (3-8, 10, 13-15, 18, 23-25, 39, 54, 55), but another view is that TonB-box movement away from the barrel wall frees the N-domain to dislodge from the channel. Next, the ligand passes through the C-domain channel (Transport). Theory and experiment suggest, but so far do not explicitly prove, that the input of energy is required at this stage. Similarly, TonB may or may not function during this phase of the transport reaction. A variety of findings raise the possibility that the N domain exits the pore during ligand uptake (35), but this idea is not fully substantiated: structural changes that facilitate ligand transport may take place in the N domain while it is resident in the channel. After transport the receptor reassembles, either by reinsertion of the N domain into the β-barrel, or by structural changes in situ within the pore, another potential phase for the input of energy and/or TonB. Lastly, the loops reopen to a state of maximum receptivity toward ligands.