Cellular\nPrion Protein Conformational Shift after\nLiquid–Liquid Phase Separation Regulated by a Polymeric Antagonist\nand Mutations

Liquid–liquid\nphase separation (LLPS) of intrinsically disordered\nproteins has been associated with neurodegenerative diseases, although\ndirect mechanisms are poorly defined. Here, we report on a maturation\nprocess for the cellular prion protein (PrP<sup>C</sup>) that involves\na conformational change after LLPS and is regulated by mutations and\npoly(4-styrenesulfonic acid-<i>co</i>-maleic acid) (PSCMA),\na molecule that has been reported to rescue Alzheimer’s disease-related\ncognitive deficits by antagonizing the interaction between PrP<sup>C</sup> and amyloid-β oligomers (Aβo). We show that PSCMA\ncan induce reentrant LLPS of PrP<sup>C</sup> and lower the saturation\nconcentration (<i>C</i><sub>sat</sub>) of PrP<sup>C</sup> by 100-fold. Regardless of the induction method, PrP<sup>C</sup> molecules subsequently undergo a maturation process to restrict\nmolecular motion in a more solid-like state. The PSCMA-induced LLPS\nof PrP<sup>C</sup> stabilizes the intermediate LLPS conformational\nstate detected by NMR, though the final matured β-sheet-rich\nstate of PrP<sup>C</sup> is indistinguishable between induction conditions.\nThe disease-associated E200 K mutation of PrP<sup>C</sup> also accelerates\nmaturation. This post-LLPS shift in protein conformation and dynamics\nis a possible mechanism of LLPS-induced neurodegeneration.