The complex of ferric-enterobactin with its transporter from Pseudomonas aeruginosa suggests a two-site model

Abstract

Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe 3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.

The structure of the iron-catecholate transporter Fiu suggests substrate import occurs via a 2-step mechanism

“…The 12 th structure, the ligand of which did not colocalize with Fe-DHB, is the enterobactin transporter PfeA from P. aeruginosa (Figure 4). In this structure the binding of enterobactin in PfeA occurs on the external face of the extracellular loops of the transporter, which entirely enclose the entrance to the lumen of the barrel [42]. It was demonstrated that PfeA binds enterobactin via 2-site binding and that the site observed in this crystal structure represents the initial substrate binding site, with the second binding site located deeper inside the transporter barrel [42].…”

“…The location of the putative external Fiu substrate binding site is conserved among TBDTs In order to validate the external substrate binding site identified in our docking analysis, we compared its location to the substrates of other TBDTs that have been structurally characterized. For this analysis we selected a 12 non-redundant TBDTs-ligand structures (Table S5) [38,[41][42][43][44][45][46][47][48][49] and superimposed them with the structure of Fiu. In 11 of these 12 structures the substrate bound in an analogous location to our Fe-DHB docking solution, with the metal ion of the respective ligands located between 2.8 and 9.5 Å from the Fe in the docked complex ( Figure 4).…”

“…In this structure the binding of enterobactin in PfeA occurs on the external face of the extracellular loops of the transporter, which entirely enclose the entrance to the lumen of the barrel [42]. It was demonstrated that PfeA binds enterobactin via 2-site binding and that the site observed in this crystal structure represents the initial substrate binding site, with the second binding site located deeper inside the transporter barrel [42]. This 2-site binding model is further supported by the structural analysis of FepA, a close homologue of PfeA, which shows that it binds enterobactin at two locations, the one of which is analogous to the PfeA binding site [50].…”

“…FepA and its closely related homologue PfeA from P. aeruginosa, initially bind enterobactin an external binding site formed by extracellular loops that entirely enclose the entrance of these transporters β-barrel [42,50]. Fiu lacks the extracellular loop structure required for this external binding site and our docking analysis suggests it binds its substrate deep in its internal cavity, at a location shared among other TBDTs [38,[41][42][43][44][45][46][47][48][49].…”

“…If the external plug loop then transitioned to an ordered state during subdomain removal by TonB, it would prevent channel formation through Fiu during import, and prevent the non-specific import of deleterious substances. Despite their significant structural differences and distinct initial binding sites, the essence of this mechanism may be shared with FepA, which possesses a second enclosed binding site deeper inside the barrel of the transporter [42,50].…”