A conformational switch governs the dual role of liquid–liquid phase separation in amyloid fibrillization

Abstract Biomolecular condensates provide dynamic environments that store and organize proteins, yet the fundamental principles determining whether condensation promotes or suppresses irreversible protein aggregation remain unclear. Here, we show that the receptor-interacting protein kinase 3 (RIPK3) RHIM domain follows distinct self-assembly pathways into condensates or amyloid fibrils depending on its conformational state. We find that predominantly folded proteins undergo liquid–liquid phase separation (LLPS), forming reversible condensates that kinetically suppress fibril formation. Partial unfolding instead promotes direct fibrillization that bypasses LLPS, whereas induced condensate formation via increasing ionic strength or addition of molecular crowder delays amyloid formation. In contrast, when the protein is predominantly unfolded, LLPS accelerates fibrillization through condensate-interface-mediated nucleation. These findings establish protein conformation as a determinant of whether condensates suppress or promote amyloid assembly, revealing a dual role for phase separation in regulating functional amyloid formation and providing a framework that connects condensate dynamics with cellular functions. Abstract Figure