Death domain-mediated higher-order oligomerization of RIPK1 drives TNF-induced signaling.

RIPK1 paradoxically orchestrates pro-survival and pro-death pathways through its scaffolding and kinase activities. Downstream of TNFR1, RIPK1 initiates the formation of two distinct multiprotein complexes: complex I for cell survival and complex IIa/IIb for apoptosis or necroptosis. However, the molecular architecture that initiates RIPK1-mediated assembly of these multiprotein signaling complexes remains unknown. Here, we elucidate the filamentous structure of the death domain of RIPK1 (RIPK1DD) at 2.5 Å resolution using cryogenic electron microscopy (cryo-EM). RIPK1DD filaments comprise three helical chains assembled via four distinct types of interactions, including a unique, hitherto undescribed Type IV interaction. Structure-guided mutagenesis of interface residues effectively disrupts RIPK1DD filament formation, impairs RIPK1 recruitment and ubiquitination within TNFR1 complex I, and abrogates TNF-induced transcriptional responses. Furthermore, death domain-mediated higher-order oligomerization drives RIPK1 autophosphorylation, which consequently licenses the assembly of complex IIa and complex IIb to execute apoptosis and necroptosis, respectively. Together, these findings reveal a critical structural basis of death domain-mediated RIPK1 oligomerization and provide fundamental mechanistic insights into RIPK1-mediated assembly of signaling complexes downstream of TNFR1.