Fig. 2: Pseudokinase domain dimerization drives full-length MLKL tetramerization in solution.
From: Phosphorylation-dependent pseudokinase domain dimerization drives full-length MLKL oligomerization
a Size-exclusion chromatograms of full-length human MLKL [red, wild-type (WT); green, T357E/S358E (TSEE) mutant] in complex with RIPK3 kinase domain, overlaid with the chromatogram of MLKL alone (black), and MLKL tetramer bound by Mb27 (blue). Fractions analysed by SDS-PAGE are labelled on the x-axis [red, MLKL (WT):RIPK3; blue, MLKL (TSEE):RIPK3]. b Stain-free reducing SDS-PAGE of full-length human MLKL:RIPK3 kinase complexes size-exclusion chromatography fractions. Data in (a, b) are representative of two independent repeats. c Mb32 (red) cannot bind the MLKL pseudokinase domain due to binding epitope overlap with the dimerization interface, while Mb27 (green) binds the dimer by binding to the ATP-binding cleft. Crystal structures of Mb32:MLKL pseudokinase domain (PDB: 7JXU; MLKL not shown) and Mb27:MLKL pseudokinase domain (PDB: 7JW7; MLKL not shown)33 were aligned with the crystal structure of p-MLKL pseudokinase domain dimer (PDB: 8SLZ; αC trace, white and slate grey) using UCSF ChimeraX. d, e Pseudokinase domain dimer is detected within p-MLKL tetramer (from co-expression with RIPK3 kinase domain), but not within the TSEE mutant MLKL:RIPK3 heterodimer. d Binding of N-terminal His6-tagged Monobodies to recombinant MLKL:RIPK3 complexes were examined by Ni-NTA precipitation and stain-free SDS-PAGE. Mb32 is unable to bind wild-type p-MLKL tetramer, confirming that pseudokinase domain dimerizes within this tetramer. Mb27 is able to bind p-MLKL tetramer, but not TSEE mutant MLKL:RIPK3 heterodimer, because RIPK3 hinders the Mb27 epitope29,33. Control Mb3359 binding to the 4HB domain is not affected by MLKL oligomeric state. Data represent two independent replicates. e Illustration of the mechanisms by which Monobodies detect pseudokinase domain dimerization in full-length MLKL complexes. Richardson (Ribbon) diagrams of Mb27 (from PDB: 7JW7)33, Mb32 (from PDB: 7JXU)33 and Mb33 (from PDB: 6UX8)59 were drawn using PyMol. f Mb27 binds exclusively MLKL tetramer upon necroptotic stimulation in HT29 cells. HT29 cells stably expressing Monobodies33 were harvested at 4.5 h post necroptosis stimulation by TSI and subjected to FLAG-immunoprecipitation. The FLAG-eluates were analysed on Blue Native (BN)-PAGE or SDS-PAGE and immunoblotting with antibodies indicated. Data representative of two independent replicates.
