MicroCal iTC200 was used for the characterization of the interaction between aTFs and small molecules. HucR and HosA were diluted in an ITC buffer [150 mM NaCl and 50 mM tris-HCl (pH 7.5)] with final concentrations of 25 and 20 μM, respectively. The small molecules UA and 4-HBA were dissolved in the same ITC buffer with a final concentration of 250 μM. The ITC conditions were as follows: 330 μl of the aTF was first injected in the sample cell. Then, the titration was initiated with a first injection of 0.4 μl of the corresponding small molecule, followed by 19 injections of 2 μl at 25°C. The blank tests were carried out by using an ITC buffer instead of aTFs in the titration step and were used for baseline correction. The instrument software was used to calculate the normalized heats released from each injection.

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