Fig. 4: Horse MLKL activation loop phosphorylation induces conformational flexibility.

a The activity of phosphomimetic mutations, S345D in rat MLKL, or T356E-S357E in horse MLKL, or alanine substitution (T356A-S357A) in horse MLKL, was tested in MLKL^−/− U937 and/or Mlkl^−/− MDF cells upon doxycycline induction of expression, in the presence (TNF, Smac mimetic, IDN-6556; TSI) and absence (untreated; UT) of necroptotic stimuli. These data are plotted alongside wild-type controls from Fig. 1. b, c The activation loop of the horse MLKL pseudokinase domain is buried in the pseudoactive site in a position occupied by ATP in conventional protein kinases, such as ERK (PDB 4GT3) (d). e Wild-type horse MLKL or alanine substitution mutants of activation loop and adjacent pseudoactive site residues were stably introduced into Mlkl^−/− MDF cells and the capacity to kill cells in the presence (TSI) or absence (UT) of necroptotic stimuli in the presence (induced) or absence (uninduced) of doxycycline-induced exogene expression quantified by PI uptake using flow cytometry. Data in a and e are shown as mean ± SEM of ≥3 independent experiments for each of two biological replicate MDF lines (n = 6 for all in a, n = 7 for T208A and Q355A-T208A, n = 8 for Y282A and Q355A) or one U937 line (n = 3). * represents statistical significance of p < 0.05 using a paired, two-tailed t-test: a wild-type horse MLKL p = 0.0017, rat MLKL S345D in MDFs UT uninduced vs UT induced p = 0.0089, rat MLKL S345D in MDFs TSI uninduced vs TSI induced p = 0.0035, rat MLKL S345D in U937s UT uninduced vs UT induced p = 0.0108 and rat MLKL S345D in U937s TSI uninduced vs TSI induced p = 0.00114. For e T208A p = 0.0013, Q355A p = 0.0099 and Q355A-T208A p = 0.0056. f A comparison of molecular dynamics simulations on horse MLKL reveals increased activation loop flexibility in the phosphorylated MLKL model. The x-axis shows residue numbers and the y-axis shows root mean square fluctuation (RMSF) across the simulation. The phosphorylated residues, pT356 and pS357, are shown in red. g A series of snapshots of phosphorylated horse MLKL show the phosphorylated activation loop moving out of the pseudoactive site. Zoomed insets show hydrogen bonds at various stages of the transition.