Electrostatic Regulation of Alpha-Synuclein Membrane Binding Couples Conformational Release to Aggregation Kinetics

Abstract

Misfolding and aggregation of α-synuclein (α-syn) are central to Parkinson’s disease (PD) pathology and are increasingly believed to be modulated by lipid-rich cellular environments. Electrostatic interactions between α-syn and anionic lipids play a key role in membrane binding and can significantly influence aggregation, yet the mechanisms by which physiologically relevant changes in membrane charge regulate the structure and dynamics of membrane-associated α-syn remain incompletely understood. Here, we investigate how membrane electrostatics reshape the conformational ensemble of membrane-bound α-syn using residue-resolved magic-angle spinning (MAS) NMR, Thioflavin T (ThT) aggregation assays, and transmission electron microscopy (TEM). Proteoliposomes made of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing 40%, 35%, or 25% anionic lipid were used to systematically vary bilayer surface charge. MAS NMR measurements show a progressive, charge-dependent redistribution of the α-syn structural ensemble. At a high 40% membrane negative charge, α-syn adopts a primarily α-helical membrane-associated conformation, where the N-terminal region and the aggregation-prone non-amyloid-β component (NAC) domain stay bound to the bilayer, while the C-terminus remains flexible. As bilayer negative charge decreases to 35% and 25% anionic lipid, electrostatic interactions weaken, and the NAC-proximal segments increasingly detach from the membrane, leading to helix-to-coil transitions and greater solvent exposure of residues that have been shown to form the core of α-syn fibrils. ThT aggregation assays, coupled with TEM analysis, reveal that these charge-dependent conformational redistributions correlate with faster primary nucleation, differences in fibril morphology, and higher-order organization. Finally, super-resolution imaging in primary neurons shows that aggregated α-syn accumulates on phosphatidic acid (PA)-enriched mitochondria-derived vesicles, providing a cellular context for the electrostatic regime examined in the model membrane system. Together, our results identify a membrane charge density of 25–35% anionic lipid range as an electrostatic transition region in which the aggregation-prone NAC domain becomes solvent-exposed while the N-terminal anchor remains membrane-associated. These findings provide a residue-level framework for how membrane electrostatics shape the conformational landscape of membrane-bound α-syn and define aggregation-competent states in lipid environments.