Necroptosis is a pro-inflammatory form of programmed cell death. Dysregulated necroptosis has been shown to play a role in a range of different human pathologies, including ischemia-reperfusion injuries and inflammatory bowel disease. The terminal effector of necroptosis is a ‘dead’ kinase, the pseudokinase MLKL (mixed lineage kinase domain-like). Upon phosphorylation by the kinase RIPK3, the cell killing activity of MLKL is activated, leading to the permeabilization of the plasma membrane to induce necroptotic cell death. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically diverge between species.
We sought to investigate the mechanism by which human RIPK3 and MLKL are regulated during necroptotic signaling. We determined the crystal structures of human MLKL pseudokinase domain in complex with two different monobodies or RIPK3 kinase domain. These structures reveal that MLKL is held in an open, inactive conformation by RIPK3 prior to necroptosis. Phosphorylation by RIPK3 drives MLKL pseudokinase domain to undergo a conformational change, which induces its dissociation from RIPK3. The closed active conformer of MLKL pseudokinase domain dimerizes via the hinge region and drives the formation of phosphorylated MLKL tetramers. This step is essential for engaging the lipid bilayer and plasma membrane lysis. Overall, this work has revealed the molecular mechanisms that regulate human MLKL and has identified novel checkpoints in the necroptosis pathway.