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. 2021 Nov 22;12:6783. doi: 10.1038/s41467-021-27032-x

Fig. 5. Bidentate C-lobe human MLKL:RIPK3 interactions govern necroptotic cell death.

Fig. 5

a Human MLKL pseudokinase domain (190–471) formed a stable complex with wild type, but not D142N and S227A, human RIPK3 (1–316) in Sf21 cells. MLKL and RIPK3 were present in the lysates for all three constructs, but only wild-type RIPK3 coeluted with MLKL from Ni-NTA resin (left) or size exclusion chromatography (right). Elution volumes of molecular weight standards are shown above chromatograms. b V385W, S317D, and T357E/S358E MLKL failed to restore death signaling in MLKL−/− HT29 cells upon treatment with the necroptotic stimulus, TSI (T, TNF; S, Smac mimetic Compound A; I, pan-caspase inhibitor IDN-6556) for 20 h, as quantified by SYTOX Green uptake using IncuCyte imaging. c I209R, V220E, L222D, S227A, A232R, V233R, and the kinase-dead D142N mutant human RIPK3 were unable to restore death signaling in RIPK3−/− HT29 cells upon treatment with the necroptotic stimulus, TSI, for 20 h. d Wild-type MLKL, but not T357E/S358E, V385W, and S417D mutants underwent phosphorylation upon 20 h TSI stimulation when expressed in MLKL−/− HT29 cells. Wild type, but not kinase-dead D142N (e), and wild-type, S227A, T224A, V220E, L222D, and A232R, but not I209R and V233R (f), RIPK3 phosphorylated MLKL upon 20 h TSI stimulation when expressed in RIPK3−/− HT29 cells. g Crucial residues for human MLKL activation by RIPK3 are shown as sticks (MLKL, gray; αC helix, yellow; activation loop, green). Zoomed panels show crucial residues in the bidentate MLKL:RIPK3 interaction centered on MLKL F386 (hydrophobic) and RIPK3 pS227 (electrostatic; dashed lines). The RIPK3 residues, I209 and V233, whose substitution with Arg blocked MLKL phosphorylation and necroptosis signaling, are shown in cyan. Death data are presented as mean ± SEM, with four independent repeats for MLKL constructs, except n = 5 for wild type, T357E/S358E and n = 3 for I242A, Q388A MLKL; and three independent repeats for RIPK3 constructs, except n = 12 for wild type, n = 8 for D142N, n = 5 for E25A, L26R, F36W, L222W, L228W, N238A, N312A, and n = 2 for R236A RIPK3. Blots are representative of three independent experiments.