α-Synuclein bound to planar membrane orchestrates self-multimerization and vesicle association

Alpha-Synuclein (α-Syn), a presynaptic protein implicated in Parkinson's disease (PD). While α-Syn-lipid interactions mediate both physiological synaptic vesicle (SV) regulation and pathological aggregation, the mechanistic divergence between wild-type (WT) and PD-associated mutants (A30P, E46K, A53T) in modulating membrane binding dynamics and SNARE-dependent vesicle function remains unresolved. Through single-molecule imaging and quantitative membrane interaction analysis, we demonstrate that α-Syn self-assembly on supported lipid bilayer (SLB) is electrostatically driven by anionic lipids (e.g., PIP 2 , PS), compared to neutral lipids (e.g., PE, cholesterol). Quantitative analysis of protein-membrane binding and single-vesicle docking revealed that α-Syn’s membrane affinity directly dictates its interference with SNARE-mediated vesicle docking. Specifically, α-Syn WT self-assembly on SLB competitively occupies synaptotagmin-1 (Syt1) binding sites on anionic lipid microdomains, suppressing vesicle docking, while pre-formed fibril showed reduced membrane binding and mild interference with vesicle docking. Moreover, both multimeric and fibrillar forms of A30P exhibited diminished membrane binding with no significant docking inhibition, whereas E46K and A53T demonstrated enhanced membrane association and docking suppression. These findings establish that α-Syn's membrane-mediated self-assembly critically determines its neurotoxic potential, with lipid-binding capacity directly correlating to synaptic dysfunction severity. Our study provides a biophysical framework for understanding how α-Syn-lipid interaction regulate pathogenic progression in PD.