Valence and Patterning of Aromatic Residues Determine the Phase Behavior of Disordered Prion-Like Domains

Prion-like domains (PLDs) are intrinsically disordered protein regions that can drive functional liquid-liquid phase separation (LLPS) in vitro and in cells. Curiously, in many neurodegenerative diseases these regions contain mutations, leading to the formation of aggregates which are strongly correlated with disease pathology. An open question reflects the underlying molecular details that explain why some sequences form dynamic reversible assemblies, while others form solid-like aggregates. Here, we use multipronged biophysical approaches that integrates across simulation, experiment, and theory to address this question and uncover the physical principles underlying how an archetypal PLD derived from hnRNPA1 avoids aggregation in favor of LLPS. The extent of compaction of individual PLDs in dilute solutions mirrors the driving forces for temperature-dependent LLPS. This compaction is directly determined by the valence of aromatic residues in PLDs. As a result, the sequence-dependent phase behaviour for PLDs can be predicted using a stickers-and-spacers model. We uncover an evolutionary preference for aromatic residues to be uniformly distributed, as opposed to being clustered, along PLD sequences. We demonstrate that this non-random patterning drives reversible LLPS whereas its disruption leads to aggregation. We conclude that the valence and patterning of aromatic residues are key determinants of LLPS in PLDs. More generally, the valence and patterning of stickers in a stickers-and-spacers model offers a quantitative approach to relate sequence features to full binodal curves.