Biochemical analysis of hSTING interaction with c-di-GMP.

<p>(A) Isothermal titration calorimetry measurement of the interaction between WT hSTING and c-di-GMP. The recombinant WT hSTING and G230A protein (C-terminal domain, CTD) were purified and quantified by a Nanodrop spectrometer. The purity of proteins was analyzed on SDS-PAGE. ITC was performed at 25 °C as described in the experimental procedures. The dissociation constants and thermodynamic parameters were measured by ITC using a MicroCal iTC200 calorimeter. (B) Overlapping ITC assay of WT and G230A mutant with c-di-GMP. Equilibrium association constant, binding stoichiometry N, enthalpy ΔH were determined by fitting the data based on a single set of identical sites model. (C) Differential scanning fluorimetry assay of WT hSTING with or without c-di-GMP. Where present, c-di-GMP was at 5 molar excess of the apo-protein. The curves represent at least two independent experiments. The ΔT_Mapp of hSTING with and without c-di-GMP was shown. The bold line denotes the WT hSTING with H₂O and the dash line is WT with c-di-GMP. (D) Thermal denaturation of G230A in the absence or presence of 5 molar excess of c-di-GMP. (E) Effects of different molar ratios of c-di-GMP on the T_Mapp of the WT or G230A hSTING in differential scanning fluorescence assay. The T_Mapp was plotted in the presence of different molar ratios of c-di-GMP. The bold line represents CTD of WT hSTING and dash line is G230A CTD. (F) Differential scanning fluorimetry assay of WT hSTING with or without 3’3’cGAMP. (G) Thermal denaturation of G230A in the absence or presence of 5 molar excess of 3’3’cGAMP. (H) Effects of different molar ratios of 3’3’cGAMP on the T_Mapp of the WT or G230A hSTING.