Table of Contents:
To ensure the supply with the essential nutrient and trace element iron, microorganisms have evolved several strategies. The secretion of low molecular weight, chelating molecules, the so called siderophores, takes a very prominent role among the bacterial iron acquisition strategies. The uptake of the iron-loaded siderophores through microorganisms constitutes a key step in this strategy and is therefore an attractive target for potential antibiotic agents. High affinity binding of siderophores is accomplished by substrate binding proteins, which are part of ATP-binding cassette transporters. The potential exploitation of the transport mechanisms through pharmacological agents demands a comprehensive functional and structural characterization of the involved proteins.
In this work an affinity chromatography-based approach for the isolation and identification of substrate binding proteins from crude cell extracts through direct interaction with its natural ligand has been established. Synthetic derivatives of the siderophore petrobactin were immobilized on an agarose−streptavidin matrix and treated with the cell lysate of a Bacillus subtilis culture, which allowed the successful identification of the petrobactin binding protein FpiA. The subsequent biochemical and physiological characterization of the protein confirmed its significance for petrobactin import and proved therefore the potential of this new method.
Furthermore, the triscatecholate binding protein FeuA from B. subtilis was structurally and functionally characterized. Cocrystal structures with the siderophores bacillibactin and enterobactin and the siderophor mimic mecam showed a nearly identical binding mode for these three related, but nevertheless distinct iron-complexanions. A basic triad within the binding pocket was identified as the main binding motif for the substrates and was examined through site-directed mutagenesis and subsequent characterization of the variants through fluorescence spectroscopy and ligand-dependent melting point analysis. Crystallographic and CD-spectroscopic experiments showed
that the binding pocket accepts solely lambda-stereoconfigured substrates, therefore the delta-stereoconfiguration of the iron-complex of enterobactin is inverted through binding by FeuA. Noticeable was the nearly identical binding mode of the protein for the different substrates, which is attended by a tilting of the two domains of FeuA of about 20°, the greatest experimentally observed domain movement of a class III substrate binding protein so far. This similarity of the binding modes implies its importance for the subsequent recognition of FeuA by the cognate transmembrane domains FeuBC of the transporter, which is facilitated through the correct positioning of conserved
glutamate residues on the surface of FeuA.
This work shows a new approach for the identification of bacterial substrate binding proteins and presents detailed structural and functional insights into the binding of triscatecholate siderophore by such proteins.