Fig. 5: Structural characterization of the TreX-TriX-TrxA tripartite complex.

a Crystal structure of TreX toxin (red) in complex with its immunity protein TriX (green) and E. coli thioredoxin TrxA (purple). b Zoom-in of the interaction site between the C-terminal residues of the TreX toxin and TrxA, highlighting a series of hydrophobic interactions and hydrogen bonds: TreX T130 forms hydrogen bond with TrxA catalytic C33 (left panel), TreX L132 forms a series of hydrophobic interactions with TrxA W32, and I61 and I76 side chains (middle panel), TreX Y137 sidechain forms hydrogen bond and hydrophobic interactions with TrxA D62 and I61, respectively (right panel). c Pulldown assays. Soluble extracts of cells producing the TreX*^ST and TrxA^H or their mutated versions were subjected to Strep affinity purification. Total extracts (T) and elutions (EL) were separated by SDS-PAGE and stained with Coomassie blue (top panel) or transferred onto nitrocellulose membrane and immunodetected with anti-Strep (middle panel) and anti-His (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. d In vitro neutralization of the toxin before or after interaction with TrxA. TreX was pre-incubated with the TriX immunity or with TrxA before adding the other components (FtsZ, biotinyl-NAD⁺, DTT) when indicated with brackets. The immunity protein was also added after the incubation of the toxin and TrxA under reducing conditions, and the reaction was continued for 30 min (right lane). e In vitro ADP-ribosylation of FtsZ with increasing amounts of TreX-TrxA complex reconstituted under denaturing conditions and purified in buffer without DTT. Reactions containing constant amount of FtsZ and biotinyl-NAD⁺ were mixed with or without the reducing agent DTT for 30 min at 37 °C. The ADP-ribosylation of FtsZ was detected by western-blot using streptavidin-alkaline phosphate conjugate.