Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS, however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similarly to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structure. Through a mutational analysis we identified three positively charged lysines in the post-octarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of liquid-liquid phase separation of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process.
Keywords: aggregation; intrinsically disordered protein; liquid-liquid phase separation; neurodegenerative disease; prion disease; prion protein; protein self-assembly.
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