Abstract
The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.
Highlights
The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense
Such divergence is consistent with an ancestral origin for the pathway in host defense, where exposure to necroptosis-interfering proteins encoded by different pathogens may have driven the co-evolution, and interspecies divergence, of RIPK3 and Mixed Lineage Kinase domain-Like (MLKL) cognate pairs[7,8,9,10,11,41,42,43,44]
The different positions of the human and mouse MLKL activation loop helices impart distinct modes of interaction with the N-lobe of human and mouse RIPK3 in their respective complexes. It is the interactions between the RIPK3:MLKL C-lobes that proved crucial for complex formation and necroptotic signaling in human cells
Summary
The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. Despite only 70% sequence identity and different autophosphorylation sites between human and mouse RIPK3 kinase domains, the C-lobes of human and mouse RIPK3 exhibited comparable topology and modes of interaction with MLKL, while the N-lobes diverged structurally and formed a distinct interface in the human RIPK3:MLKL complex. These differences could be attributed to human MLKL assuming an open conformation with a helical activation loop that displaces the key regulatory element, the αC helix. The critical functions of these residues in human RIPK3 and MLKL pinpoint the interface between their C-lobes as the crucial determinant of RIPK3:MLKL selective recognition across species
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