The structure of the bacterial iron-catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism

Abstract

The ferric iron uptake (Fiu) transporter from Escherichia coli functions in the transport of iron-catecholate complexes across the bacterial outer membrane, providing the bacterium with iron, which is essential for growth. Recently it has become clear that Fiu also represents a liability for E. coli because its activity allows import of antimicrobial compounds that mimic catecholate. This inadvertent import suggests the potential utility of antimicrobial catechol siderophore mimetics in managing bacterial infections. However, to fully exploit these compounds, a detailed understanding of the mechanism of transport through Fiu and related transporters is required. To address this question, we determined the crystal structure of Fiu at 2.1-2.9 Å and analyzed its function in E. coli Through analysis of the Fiuo crystal structure, in combination with in silico docking and mutagenesis, we provide insight into how Fiu and related transporters bind catecholate in a surface-exposed cavity. Moreover, through determination of the structure of Fiu in multiple crystal states, we revealed the presence of a large, selectively gated cavity in the interior of this transporter. This chamber is large enough to accommodate the Fiu substrate and may allow import of substrates via a two-step mechanism. This would avoid channel formation through the transporter and inadvertent import of toxic molecules. As Fiu and its homologs are the targets of substrate-mimicking antibiotics, these results may assist in the development of these compounds.

Keywords: Escherichia coli (E. coli); TonB-dependent transporter; X-ray crystallography; bacterial outer membrane; ferric iron uptake (Fiu); iron acquisition; membrane transport; outer membrane; protein structure; siderophore; solute uptake.

Figure 1.

Fiu belongs to a distinct group of catecholate siderophore transporters. A, a phylogenetic tree of diverse, functionally characterized TBDTs, showing that Fiu forms a clade with PiuA/D that is distant from the catecholate siderophore transporters FepA and Cir. Catecholate-transporting TBDT subgroups are color coded: blue, enterobactin-transporting; green, nonenterobactin-transporting FepA-related; red, nonenterobactin-transporting Fiu-related. Circles represent TBDTs present in E. coli BW25113. B, a scheme showing the sequential deletion of TBDTs in E. coli BW25113 utilized in this study, colored as in A. C, strains from B grown on LB agar in the presence of 0–150 μm 2,2′-bipyridine (BP). Sequential loss of FepA and Fiu leads to defects in the ability of strains to grow under iron-limiting conditions.

Figure 2.

The crystal structure of Fiu reveals a large gated internal cavity. A, the crystal structure of fully ordered Fiu (crystal state 2), shown as a cartoon representation with rainbow colors running from N-terminal (blue) to C-terminal (red). B, cutaway outline representation of Fiu crystal structures, showing the internal cavity selectively occluded in crystal state 2 as well as the effect of removal of the labile subdomain of the TBDT plug on Fiu channel formation through the membrane. C, composite cutaway view of Fiu, showing the N-terminal plug domain as a cartoon, with the variably ordered plug loop (blue) and labile plug subdomain (red) highlighted. Outer membrane is abbreviated to OM in this figure.

Figure 3.

Extracellular loop stability in crystal states of Fiu and PiuA/PiuD. A, stereo cartoon view of Fiu in crystal states 1 and 2, illustrating the variably ordered extracellular barrel loops 7, 8, and 9 (L7–L9) and the plug domain loop (PL). Loops are color-coded as follows: L7, salmon; L8, red; L9, brick red; plug domain loop, blue. Loop termini are shown as spheres where the loop is disordered. B, PiuA and PiuD crystal structures from P. aeruginosa and A. baumannii presented as for Fiu in A. The crystal structures of PiuA and PiuD display an analogous pattern of loop order/disorder to that observed for Fiu. C, cutaway outline representation of PiuA and PiuD, showing the presence of a selectively gated internal cavity. Outer membrane is abbreviated to OM in this figure.

Figure 4.

Top-ranked docking solutions between Fiu and the Fe–DHB substrate. A, the location of the top-ranked docking modes in a cartoon representation of Fiu, for docking run 1 (R1M1), which includes the entire extracellular portion of the transporter, and docking run 2 (R2M1), in which the internal cavity is excluded from the search area. B, the same docking solutions as in A, with a cutaway composite view of Fiu.

Figure 5.

The location of the Fiu putative external Fe–DHB binding site compared with other TBDT substrate complexes. A, the location of the Fe–DHB complex docked with Fiu compared with that of other substrates bound to in the crystal structures of superimposed TBDTs. Fiu is shown as a cartoon rainbow and in the same view as a white surface representation below. The location of the metal centers of different TBDT substrates are shown as colored spheres and labeled. B, the Fiu Fe-DHB docked complex shown as in A but in a different orientation. Representative distances between Fe–DHB and the TBDT substrate metal ions are shown, colored as in A.

Figure 6.

The effect of mutations in the Fiu external substrate-binding site on the function of Fiu in vivo. A, bar graph indicating the effect of Fiu binding site mutations of the ability of pBAD24Fiu to complement E. coli BW25113 Δ6. The length of the bar graph indicates the maximum concentration of 2,2′-bipyridine at which growth of the complemented strain was observed on solid LB agar; experimental data are shown in Fig. S5. B, stick and cartoon representation of the Fiu extracellular loops, showing the location of the residues in the putative substrate-binding site subjected to mutagenesis. C, magnified view of the Fiu substrate-binding site shown in B, rendered as a surface model with mutated residues labeled (left) and Fe–DHB shown (right). D, stereo image showing the residues mutated in the Fiu external binding site as a stick representation. The Fe–DHB complex is shown as a line and sphere representation for DHB and Fe³⁺, respectively. Colors are consistent through all panels and indicate the effect of mutagenesis on Fiu function: brick red, inactivation or significant defect in Fiu function; pink, minor defection in Fiu function; green, no defect in Fiu function.