Pathological Mutation E46K Acts as an Electrostatic Switch to Increase Distant Interdomain Interactions and Promote α‑Synuclein Phase Separation.

The Parkinson's disease-related protein α-synuclein can form solid amyloid fibrils through liquid-liquid phase separation (LLPS) and liquid-to-solid phase transition. The most deleterious familial mutation E46K has recently been shown to enhance α-synuclein LLPS and subsequent solidification; yet, the precise mechanisms remain largely unknown. Here, using molecular dynamics simulations at different spatiotemporal scales combined with biochemical experiments, we show that the E46K mutation acts as an electrostatic switch to remodel interactions between the oppositely charged N-terminal domain (NTD) and C-terminal domain (CTD) of α-synuclein. This remodeling shifts the interaction site of NTD with CTD from its N-terminus to the mutation region. Such reorganization increases the hydrophobic solvent exposure of key LLPS-promoting motifs, potentially facilitating intermolecular interactions that initiate phase separation. Phase coexistence simulations further support this hypothesis, showing that the E46K mutant exhibits enhanced LLPS and solidification propensities. This enhancement is primarily driven by intermolecular electrostatic interactions between the mutation region and the CTD, followed by the hydrophobic NAC-NAC interactions mediated by the LLPS-promoting motifs. Collectively, these results reveal that NTD-CTD electrostatic crosstalk acts as the key modulator of α-synuclein phase separation, while NAC-NAC interactions play an auxiliary role, both of which synergistically govern α-synuclein phase separation. This study offers a complete and detailed mechanistic framework for understanding α-synuclein phase separation and its enhancement induced by the E46K mutation.