LAUSR

Significance Necroptosis is an emergency controlled cell-death mechanism, which only may be activated in case of impaired apoptosis. Necroptosis leads to unchecked release of proinflammatory cytoplasmatic content of the cells and is thereby implicated in disease. Two covalent inhibitor classes targeting the same cysteine of the necroptosis effector protein mixed-lineage kinase domain-like protein (MLKL) are reported. Here, we unravel the mode of action of the xanthine class of inhibitors that work by stabilizing the inactive state of MLKL by an essential π–π stacking interaction. The widely used covalent tool compound Necrosulfonamide (NSA) employs a distinct mode of action. As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.