Cellular Prion Protein Conformational Shift after Liquid–Liquid Phase Separation Regulated by a Polymeric Antagonist and Mutations

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins has been associated with neurodegenerative diseases, although direct mechanisms are poorly defined. Here, we report on a maturation process for the cellular prion protein (PrP^C) that involves a conformational change after LLPS and is regulated by mutations and poly(4-styrenesulfonic acid-<i>co</i>-maleic acid) (PSCMA), a molecule that has been reported to rescue Alzheimer's disease-related cognitive deficits by antagonizing the interaction between PrP^C and amyloid-β oligomers (Aβo). We show that PSCMA can induce reentrant LLPS of PrP^C and lower the saturation concentration (<i>C</i>_sat) of PrP^C by 100-fold. Regardless of the induction method, PrP^C molecules subsequently undergo a maturation process to restrict molecular motion in a more solid-like state. The PSCMA-induced LLPS of PrP^C stabilizes the intermediate LLPS conformational state detected by NMR, though the final matured β-sheet-rich state of PrP^C is indistinguishable between induction conditions. The disease-associated E200 K mutation of PrP^C also accelerates maturation. This post-LLPS shift in protein conformation and dynamics is a possible mechanism of LLPS-induced neurodegeneration.