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Amyloid fibrils can form biologically relevant functional assemblies. The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) orchestrate the formation of amyloid-like fibrils essential for propagating cell death signals. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been elucidated, the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive. We present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy. The fibrils adopt an N-shaped fold consisting of three β-sheets stabilized by hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the β2-β3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. These findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids. This study reveals the atomic structure of RIPK1 fibrils, a type of physiological amyloid that helps control inflammation and regulated cell death in human cells.

Receptor-interacting protein kinase 3 (RIPK3) drives necroptosis by assembling into functional amyloid fibrils. Here we show that lipids modulate RIPK3 amyloidogenesis by stabilizing an aggregation-prone intermediate. While electrostatic repulsion maintains RIPK3 in a soluble state, charge compensation alone is not sufficient for fibril formation and hydrophobic contacts are required to initiate nucleation. Using solution-state NMR, fluorescence-based assays and polymer-encased lipid particles, we demonstrate that negatively charged membranes selectively recruit RIPK3 and restrict its conformational flexibility, accelerating aggregation. These findings reveal a membrane-guided mechanism for RIPK3 assembly and suggest that lipid surfaces, like those implicated in pathological amyloid formation, may modulate functional amyloidogenesis even in the absence of canonical necroptotic stimuli. The receptor-interacting protein kinase 3 (RIPK3) drives necroptosis by assembling into functional amyloid fibrils, but its amyloidogenesis remains underexplored. Here, the authors show that negatively charged membrane mimetics modulate this process by stabilizing a RIPK3 aggregation-prone intermediate conformation, accelerating fibril assembly even in the absence of necroptotic stimuli.

Amyloid fibrils, typically associated with neurodegenerative diseases, also play critical roles as functional assemblies in biological processes. The RIP homotypic interaction motifs (RHIMs) in receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) are essential for necroptosis, orchestrating the formation of amyloid-like fibrils that assemble into necrosomes. These supramolecular complexes propagate cell death signals and activate effectors like MLKL. While the structures of human RIPK3 (hRIPK3) homomeric fibrils and RIPK1-RIPK3 heteromeric fibrils have been resolved, the atomic structure of human RIPK1 (hRIPK1) homomeric fibrils has remained elusive. Here, we present a high-resolution structure of hRIPK1 RHIM-mediated amyloid fibrils, determined using an integrative approach combining cryoprobe-detected solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy. The fibrils adopt an N-shaped amyloid fold consisting of three β-sheets stabilized by the conserved IQIG RHIM motif through hydrophobic interactions and hydrogen bonding. A key hydrogen bond between N545 and G542 closes the β2-β3 loop, resulting in denser side-chain packing compared to hRIPK3 homomeric fibrils. This structural feature likely contributes to the compact architecture of hRIPK1 fibrils, in contrast to the more relaxed S-shaped fold observed in hRIPK3. These findings provide structural insights into how hRIPK1 homomeric fibrils nucleate hRIPK3 recruitment and fibrillization during necroptosis, offering broader perspectives on the molecular principles governing RHIM-mediated amyloid assembly and functional amyloids.Competing Interest StatementThe authors have declared no competing interest.