Fig. 4

Molecular analysis of ChAD binding to Mtb-SecB^TA. a ITC profile of ChAD titration into Mtb-SecB^TA (upper and lower parts represent raw and integrated binding heats, respectively). b Differences in normalized fluorescence generated by various concentrations of ChAD added to Mtb-SecB^TA during MST analysis and calculated fit. Error bars are as calculated with the NanoTemper analysis software based on independent experimental data. c Experimental SAXS data for Mtb-SecB^TA in the unbound state (red line), in the presence of ChAD (green line), and for the complex formed with Mtb-HigA1 (blue line) and comparison with the theoretical scattering patterns computed from the Mtb-SecB^TA/ChAD structure (black line). The inset shows the SAXS-derived Kratky plots. d Deconvoluted native mass spectrum of Mtb-SecB^TA incubated with 4 molar equivalents of ChAD peptide. The species detected correspond to the Mtb-SecB^TA tetramer (MW 81,575 Da, purple circle) and to the binding of up to four ChAD peptides with MWs of 83,157; 84,746; 86,334 and 87,922 (cyan, green, orange, and red, respectively). e Native MS-derived relative abundance of the different species obtained upon titration with 8 molar equivalents of the ChAD peptide and other 13-mer peptides evaluated in this study (peptide numbering is according to ref. ²⁹)