Fig. 3 | Nature Communications

Fig. 3

From: Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis

Fig. 3

hMLKL(2–471) tetramerizes in a daisy-chain arrangement. a Liposome dye release assay of K331N hMLKL(2–471) at 1 μM in the absence (blue) and presence (red) of 500 μM ATP. Dye release was monitored by spectrophotometry at 485 nm over 30 min. Data represent mean ± SEM of three independent assays. b Small angle scattering profile of averaged and background subtracted data from the apex of inline size exclusion peak (black circles). Agreement between the experimental and theoretical scatter (red circles) calculated from a rigid body tetramer model in SASREF is reflected in the χ value of 0.54. Inset: Guinier plot of experimental SAXS data for q.Rg < 1.3, illustrating that aggregated species do not contribute substantively to the scatter. c Interatomic distance distribution plot, P(r), profile calculated from scattering data with GNOM. Maximum particle dimension, Dmax, was estimated as 170 Å. d Arrangement of subunits within the hMLKL tetramer. e Electrostatic surface of one subunit in the tetramer. Charge distribution is consistent with available presentation of residues with positive charge in the 4HB domain to engage lipid (lipid-binding residues defined in ref. 17 are labeled). f Model of tetramer binding to lipid bilayer, where the planar presentation of the four N-terminal domains suggests simultaneous engagement. g, h Differential hydrogen–deuterium exchange (wild-type vs E351K hMLKL, 5 min time point) for each analyzed peptide was averaged across overlapping sequences for each amino acid residue (excluding the first three residues of each peptide due to high rates of back exchange) and mapped onto the crosslink-refined E351K hMLKL monomer model (g) or the hMLKL tetramer model shown with 4HB down (left) or PsK down (right; h). Dark gray coloring represents regions of the protein excluded from analysis owing to insufficient sequence coverage

Back to article page