Fig. 2. MenA₁ induces phosphorylation of MenT₁, resulting in a heterogenous toxin population.

a Phos-Tag SDS-PAGE of purified MenT₁ samples either expressed in the absence and presence of MenA₁, or co-incubated in the absence or presence of combinations of MenA₁, MgCl₂, and CTP. b Densitometric analysis of bands visualized by Phos-Tag SDS-PAGE. Data are representative of three independent biological replicates and bars represent the mean +/- SEM. c Crystal structure of the MenA₁:MenT₁ complex (PDB 8AN5) shown as a cartoon and coloured orange (MenT₁α), blue (MenA₁), and light orange (MenT₁β). MenT₁ conserved active site residues are shown as sticks for reference. d Close-up view of the boxed region in (c), rotated to display MenA₁ vertically, with residues partaking in hydrophobic interactions shown for reference. e Phos-Tag SDS-PAGE of purified MenT₁ incubated with MgCl₂ and CTP in the absence or presence of either wild-type MenA₁, or N1-32 and L14R/V19R mutants. f Densitometric analysis of (e) reveals phosphorylation activity is localized to the MenA₁ N-terminal α-helix, with L14R/V19R mutations inhibiting phosphorylation. Data are representative of two independent biological replicates. g Cartoon of MenA₁ with residues of interest shown as sticks. Source data are provided as a Source Data file.