Fig. 1: RIPK3-mediated phosphorylation drives human MLKL pseudokinase domain dimerization by promoting the closed conformation.
From: Phosphorylation-dependent pseudokinase domain dimerization drives full-length MLKL oligomerization
a Human MLKL pseudokinase domain (grey ribbon; yellow, αC helix; green, activation loop) undergoes a conformational change upon phosphorylation of the activation loop (T357/S358). Left, crystal structure of p-MLKL pseudokinase domain adopts a closed conformation with an intact VAIK-αC salt bridge (zoomed panel) and disordered activation loop. Right, previously published structure of MLKL pseudokinase domain in complex with RIPK3 kinase domain29 adopts an open conformation with a disrupted VAIK-αC salt bridge (zoomed panel) and a structured, helical activation loop where T357/S358 are not phosphorylated. Zoomed panel, unphosphorylated S358 is buried underneath the αC helix (orange transparent surface) and forms a hydrogen bond with T246 of the αC helix. b Human phosphorylated (p)-MLKL pseudokinase domain dimer model generated from the crystallographic symmetry mate (symmetry mate coloured slate grey). c Small angle X-ray scattering (SAXS) scattering profile (grey points) of human p-MLKL pseudokinase domain support the pseudokinase domain dimer model. Theoretical scattering profile of p-MLKL pseudokinase domain dimer was calculated using CRYSOL and shown as red line. Guinier plot is shown as the insert. Standardized residual plot comparing theoretical and experimental scattering profiles is shown on the bottom. d Mb27 (teal and salmon) binding stabilizes the closed MLKL pseudokinase domain (grey ribbon; yellow, αC helix; green, activation loop) conformation and promotes pseudokinase domain dimerization in solution. Model of MLKL:Mb27 pseudokinase domain complex in a 2:2 stoichiometry generated from the symmetry mate of MLKL:Mb27 co-crystal structure (PDB: 7JW7; symmetry mate of MLKL coloured slate grey)33. e Mb32 (teal and salmon) binding stabilizes the open MLKL pseudokinase domain conformation and stabilizes monomeric pseudokinase domain in solution. Crystal structure of Mb32:MLKL pseudokinase domain heterodimeric complex (PDB: 7JXU)33. f, g SAXS scattering profiles (grey points) of Monobody:MLKL complexes (c MLKL:Mb27; d MLKL:Mb32) show strong concordance with the theoretical scatter (red) calculated from their respective complex models. The theoretical scattering curves from (f, g) are included for comparison in (g, f), respectively, as blue dashed lines.
