Zinc ions trigger the prion protein liquid-liquid phase separation

Abstract

Prion diseases are characterized by the misfolding and conversion of the monomeric prion protein (PrP) to a multimeric aggregated pathogenic form, known as PrP^Sc. We and others have recently shown that biomolecular condensates formed via liquid-liquid phase separation of PrP can undergo maturation to solid-like species that resemble pathological aggregates, and this process is modulated by DNA, RNA, and oxidative conditions. Conversely, the most well-studied ligand of PrP, copper ions, induce liquid-like condensates of PrP that accumulate Cu²⁺ in vitro, and live PrP^C-expressing cells show condensation at the cell surface as triggered by physiologically relevant conditions of Cu²⁺ and protein concentrations. Since PrP can also bind to Zn²⁺ through its intrinsically disordered N-terminal domain, though with different affinities and binding modes than Cu²⁺, we hypothesized that Zn²⁺ could modulate PrP phase separation differently from copper ions. Using an appropriate buffer with negligible metal ion binding, as well as relevant pH, ionic strength, molecular crowding, and Zn²⁺ concentrations, we show that recombinant PrP undergoes phase separation with Zn²⁺. Furthermore, we show that metal ion-induced condensation of PrP is dependent on the N-terminal domain (residues 23–90). In vitro Fluorescence Recovery After Photobleaching (FRAP) experiments and thioflavin T aggregation kinetics support key differences in the molecular properties of PrP:Zn²⁺ versus PrP:Cu²⁺ phase separated states. FRAP analysis indicated that both Cu²⁺ and Zn²⁺ promote liquid-like PrP condensates; however, PrP:Zn²⁺condensates exhibit a faster recovery. Cu²⁺ pronouncedly inhibits seed-induced PrP misfolding, whereas Zn²⁺ provides a milder delay in PrP aggregation. Our findings provide insights on Zn²⁺-induced phase separation of PrP, supporting a variety of previously proposed functions of PrP in metal sequestering and uptake, processes that could be effectively regulated through biomolecular condensation.