Fig. 2 | Nature Communications

Fig. 2

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

Fig. 2

4HB packs against the αC helix in the hMLKL PsKD and is displaced upon ATP binding. a E351K hMLKL(2–471) structure modeled using molecular dynamics based on distance restraints defined by DMTMM crosslinking experiments (Supplementary Fig. 2a, Supplementary Data 1). The αC helix is highlighted in green, while α3 and α4 of the 4HB domain (yellow) pack against the αC helix. The RIPK3 substrates, Thr357 and Ser358, are shown as sticks. b Mirror plot demonstrating deuteration (percentage of theoretical maximum) of E351K hMLKL peptides±ATP over the indicated time points. Error bars represent SEM of triplicate experiments. Each tick on the x axis represents an individual peptide. Overlapping or adjacent peptides were identified for all regions of the protein except for a small section of the N-terminal 4HB. A line break in this region has been introduced to illustrate the gap in sequence coverage (see Supplementary Fig. 3a for peptide sequence coverage). Overlapping regions of peptide coverage are illustrated using tiled lines below the x axis. All SEM values did not exceed ±5.96 from n = 3 independent time course experiments; error bars omitted for clarity. c Differential deuterium uptake of E351K hMLKL peptides ±ATP over the indicated time points (calculated + ATP deuteration minus -ATP deuteration). As in b, each tick represents an individual peptide with a line break introduced to illustrate a gap in sequence coverage. Peptides corresponding to overlapping sequences are shown as tiles below the plot. The corresponding regions of hMLKL the peptides map to are shown schematically between c and d, with sequence elements that undergo differential exchange±ATP highlighted in blue. d Differential hydrogen–deuterium exchange (E351K hMLKL±ATP, 60 min time point) for each analyzed peptide was averaged across overlapping sequences per amino acid residue (excluding the first 2 residues of each peptide due to high rates of back exchange) and mapped onto the E351K hMLKL model (see Supplementary Fig. 3b). Dark gray coloring represents regions of the protein excluded from analysis owing to insufficient sequence coverage

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