Dissecting the Molecular Determinants of α‐synuclein Phase Separation and Condensate Aging: The Pivotal Role of β‐Sheet‐Rich Motifs

Abstract Emerging evidence indicates that liquid‐liquid phase separation of α‐synuclein occurs during the nucleation step of its aggregation, a pivotal step in the onset of Parkinson's disease. Elucidating the molecular determinants governing this process is essential for understanding the pathological mechanisms of diseases and developing therapeutic strategies that target early‐stage aggregation. While previous studies have identified residues critical for α‐synuclein amyloid formation, the key residues and molecular drivers of its phase separation remain largely unexplored. Herein, multiscale simulations and experimental approaches are employed to uncover the molecular determinants dictating α‐synuclein phase separation and the pre‐solidification of its condensates. Seven motifs are identified that exhibit high β‐sheet propensity in the monomeric state of α‐synuclein and progressively increase in β‐sheet content during condensation. Notably, two C‐terminal motifs engage in a percolated network of intermolecular interactions through transient hydrogen bonds, contributing to the phase boundary properties. Deletion of these motifs reduces the phase separation ability of α‐synuclein, underscoring their essential roles in this process. Together, the findings reveal crucial phase separation hotspots and shed light on the molecular mechanism underlying α‐synuclein phase separation, offering significant insights and novel potential therapeutic targets for Parkinson's disease.