Proc. Natl. Acad. Sci. USA 10.1073/pnas.1408987111

Credit: PNAS

The induction of necroptosis requires receptor interacting protein kinase-3 (RIPK3)-mediated phosphorylation of the activation loop in the pseudokinase mixed lineage kinase domain-like (MLKL). MLKL phosphorylation has been proposed to lead to a conformational change in the pseudokinase domain that promotes MLKL oligomerization and plasma membrane localization, thereby activating downstream cell death effectors. To determine the MLKL domains required for cell death, Hildebrand et al. expressed MLKL fragments in mlkl-deficient fibroblasts under control and necroptosis-induced conditions. Expression of the pseudokinase domain was sufficient to block necroptosis, whereas expression of the N-terminal four-helix bundle (4HB) domain promoted constitutive necroptosis. Consistent with the importance of the 4HB domain, alanine scanning mutagenesis revealed two clusters in 4HB critical for the function of MLKL; mutations in cluster 1 prevented MLKL oligomerization and membrane localization, whereas mutations in cluster 2 exhibited normal membrane localization but were unable to initiate cell death. The authors proposed that 4HB is normally constrained by the pseudokinase domain and that RIPK3-induced conformational changes in the pseudokinase domain may free the 4HB domain to promote cell death. To test their model, the authors identified a small molecule that binds and disrupts the pseudokinase domain. Treatment with this small molecule prevented membrane localization and necroptotic cell death, suggesting that coordination between the pseudokinase and 4HB domains may be essential to control entry into necroptosis.