Figure 2: Assays for specific interaction between human LCN2 protein and c-di-GMP.

(a) Cross-linking assay. STING was used as a positive control (lane 1). A competitive experiment was carried out by addition of unlabeled c-di-GMP at 67-, 134- and 333-fold excess to the reaction mixtures containing 15 μM rLCN2 and 1.5 μM c-di-[³²P]GMP (lanes 2– 4). [³²P]GTP was used as a negative control molecule. The reaction samples were subjected to a 12% w/v SDS–PAGE and radioactive gels were exposed to a storage phosphor screen (GE Healthcare). Radiolabelled nucleotides is indicated by an asterisk (*). (b) ITC assays for the specific interaction between rLCN2 and c-di-GMP. Original titration data and integrated heat measurements are shown in the upper and lower plots, respectively. Open and filled rectangles indicate data for c-di-AMP and c-di-GMP, respectively. The solid line in the bottom panel represents the best fit to a one-site binding model of the interaction of rLCN2 with c-di-GMP. c-di-AMP was used as a control. (c) Schematic of the predicted contacts between c-di-GMP and LCN2 from docking results. Potential hydrogen bonds are indicated as green dashed lines. (d) Cross-linking assays for the binding of c-di-GMP with five recombinant mutant LCN2 proteins. Two mutant proteins, rLCN2-W79A (lane 3) and rLCN2-K125A (lane 4), did not exhibit c-di-GMP binding activities. (e) Assays for c-di-GMP secretion by two bacterial strains. The concentration of extracelluar release of c-di-GMP was obtained, 1.042 μM for E. coli BL21(DE3) and 0.568 μM for M. tuberculosis H37Ra. Error bars represent the variant range of the data derived from three biological replicates.