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The Escherichia coli Fec system, consisting of an outer membrane receptor (FecA), a periplasmic substrate binding protein (FecB) and an inner membrane permease-ATPase type transporter (FecC/D), plays an important role in the uptake and transport of Fe(3+)-citrate. Although several FecB sequences from various organisms have been reported, there are no biophysical or structural data available for this protein to date. In this work, using isothermal titration calorimetry (ITC), we report for the first time the ability of FecB to bind different species of Fe(3+)-citrate as well as other citrate complexes with trivalent (Ga(3+), Al(3+), Sc(3+) and In(3+)) and a representative divalent metal ion (Mg(2+)) with low μM affinity. Interestingly, ITC experiments with various iron-free di- and tricarboxylic acids show that FecB can bind tricarboxylates with μM affinity but not biologically relevant dicarboxylates. The ability of FecB to bind with metal-free citrate is also observed in (1)H,(15)N HSQC-NMR titration experiments reported here at two different pH values. Further, differential scanning calorimetry (DSC) experiments indicate that the ligand-bound form of FecB has greater thermal stability than ligand-free FecB under all pH and ligand conditions tested, which is consistent with the idea of domain closure subsequent to ligand binding for this type of periplasmic binding proteins.

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP-cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L-histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L-His and many related naturally occurring compounds. Our data confirm that L-His is the preferred ligand, but that 1-methyl-L-His and 3-methyl-L-His can also bind, while the dipeptide carnosine binds weakly and D-histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L-Arg, homo-L-Arg, and post-translationally modified methylated Arg-analogs also bind with reasonable avidity, with the exception of symmetric dimethylated-L-Arg. In contrast, L-Lys and L-Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein-based reagentless biosensor.

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