Fig. 4: The dCACHE domain of PctA prefers the nonborated form of AI-2.

a, b Higher binding affinity of the products of the Pfs/LuxS reaction to PctA-LBD is detected under borate-depleted conditions. The binding affinity was evaluated using ITC analysis. In vitro reaction of SAH with Pfs and LuxS proteins was performed with plasticware and borate-depleted water, and concentration of DPD/AI-2 in the reaction products is ~13 μM. Proteins were dialyzed against borate-depleted buffer before use. ITC data and plots of injected heat for automatic injections of the borate-depleted reaction products (a) or the borate-depleted products supplemented with 150 μM boric acid (b) into the sample cell containing 1.3 μM borate-depleted PctA-LBD (a) or borate-depleted PctA-LBD supplemented with 150 μM boric acid (b) are shown in the upper and lower plots, respectively. Microcalorimetric data were corrected by subtracting the heats of dilution for ligand solutions injected into buffer (Supplementary Fig. 15b, c). Three independent ITC experiments were performed and similar results were obtained. The K_d and binding stoichiometry (n) calculated by the NanoAnalyze software from three independent experiments are presented as mean ± s.d. c Predicted binding mode of R-THMF in the amino acid-binding pocket of PctA-LBD. PctA-LBD (PDB ID: 5T7M)²⁶ and R-THMF extracted from R-THMF-LsrB (PDB ID: 1TJY)⁷ were used for Glide XP docking analysis. The conformation with the lowest docking energy is given via the Chimera software. The key residues of PctA-LBD involved in R-THMF binding are shown as purple sticks, and R-THMF is shown as cyan sticks. Five potential hydrogen bonds are indicated by dashed lines. d Binding of DPD/AI-2 (700 μM) to PctA-LBD and its mutants (70 μM) under normal conditions. The binding affinity was evaluated using ITC analysis. Data shown are mean  ± s.d. of three biological replicates. WT, wild-type.