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Janine Kamps, Verian Bader, Konstanze Winklhofer, Jörg Tatzelt

• Liquid-liquid phase separation (LLPS) of the mammalian prion protein is mainly driven by its intrinsically disordered N-terminal domain (N-PrP). However, the specific intermolecular interactions that promote LLPS remain largely unknown. Here, we used extensive \(\underline {mutagenesis}\) and comparative analyses of evolutionarily distant PrP species to gain insight into the relationship between \(\underline {protein}\) \(\underline {sequence}\) and phase behavior. LLPS of mouse PrP is dependent on two polybasic motifs in N-PrP that are conserved in all \(\underline {tetrapods}\). A unique feature of mammalian N-PrP is the octarepeat domain with four \(\underline {histidines}\) that mediate binding to \(\underline {copper ions}\). We now show that the octarepeat is critical for promoting LLPS and preventing the formation of PrP aggregates. Amphibian N-PrP, which contains the polybasic motifs but lacks a repeat domain and histidines, does not undergo LLPS and forms nondynamic protein assemblies indicative of aggregates. Insertion of the mouse octarepeat domain restored LLPS of amphibian N-PrP, supporting its essential role in regulating the phase transition of PrP. This activity of the octarepeat domain was neither dependent on the four highly conserved histidines nor on copper binding. Instead, the regularly spaced \(\underline {tryptophan}\) residues were critical for regulating LLPS, presumably \(\it via\) cation–\(\pi\) interactions with the polybasic motifs. Our study reveals a novel role for the tryptophan residues in the octarepeat in controlling phase transition of PrP and indicates that the ability of mammalian PrP to undergo LLPS has evolved with the octarepeat in the intrinsically disordered domain but independently of the histidines.