Fig. 4: Mutation of MLKL R292 and T374 in the Mb27 binding epitope inhibits necroptosis.

a Sequence alignment of human and mouse MLKL pseudokinase domains. Residues of mouse MLKL that interact with RIPK3 in the MLKL:RIPK3 complex structure (PDB accession 4M69⁴⁷; as predicted by PDBePISA⁴⁶) in red text; key structural elements in teal; activation loop residues phosphorylated by RIPK3 in green. b The mouse MLKL pseudokinase ___domain (cyan):RIPK3 kinase ___domain (purple) co-crystal structure (PDB accession 4M69)⁴⁷ with non-conserved residues in human MLKL and RIPK3 in red and yellow, respectively. c, d The human MLKL:Mb27 structure was superimposed on the mouse MLKL:RIPK3 complex structure (PDB 4M69), with Mb27-proximal residues identified for characterization in e shown as sticks (c). d The Mb27-binding interface on the human MLKL pseudokinase (cyan surface), residues within 4.5 Å of the superimposed mouse RIPK3 kinase ___domain (PDB 4M69) (purple surface) and overlapped residues in the Mb27- and mouse RIPK3-epitopes, V371, K372, S373, and S417 (orange surface) are shown. The αC helix (yellow), and previously implicated RIPK3-interacting regions of human MLKL, the activation loop (green) and F386 (blue sticks)^17,35, and T374 implicated herein, are highlighted. e Evaluation of necroptotic signaling by wild-type and Mb27 binding epitope mutants of full-length human MLKL in MLKL^−/− HT29 cells. Wild-type or mutant human MLKL expression was induced with doxycycline (Dox) and cell death was measured by SYTOX Green uptake (1/mm²) quantified using IncuCyte S3 live cell imaging in the presence or absence of a necroptotic stimulus (TNF, Smac mimetic, IDN-6556; TSI) for 20 h. Two independent cell lines were generated for WT, S239A, Q236A, and one for other MLKL mutants; WT lines were assayed in n = 5 independent experiments. TSEE, R333A and one independent Q236A line were assayed in n = 4 independent experiments; other mutants in n = 3 independent experiments. Data are plotted as mean ± SEM. f Alanine substitution of MLKL R292 likely disrupts key interactions with neighboring residues. g Wild-type and R292A, but not T374D, human MLKL expressed in MLKL^−/− HT29 cells were phosphorylated in response to 3 h TSI treatment. Data are representative of duplicate independent experiments.