In dopaminergic neurons, a-synuclein (αS) partitions between a disordered cytosolic state and a lipidbound state. Binding of αS to membrane phospholipids is implicated in its functional role of synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free and lipid-bound αS state, and between the different bound states, is slow on the NMR timescale, being in the range of 1-10 s −1 . Partitioning of the binding modes is dependent on the lipid: αS stoichiometry, and tight binding with slow exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the Nterminus of αS adopts an α-helical conformation while succeeding residues retain characteristics of a random coil. The C-terminal 40 residues remain dynamically disordered, even at high lipid concentration, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamical properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide NOE buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr 39 indicates an ordered environment for this "dark state." Titration of αS with increasing amounts of lipids suggests that the binding mode under high lipid conditions remains qualitatively similar to the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface, and instead suggest that considerable remodeling of the vesicles is induced by αS.

Three familial variants of the presynaptic protein α-synuclein (αS), A30P, E46K, and A53T, correlate with rare inherited Parkinson’s disease (PD), while wild-type αS is implicated in sporadic PD. The classic manifestation of both familiar and sporadic PD is the formation of fibrillar structures of αS which accumulate as the main component in intraneuronal Lewy bodies. At presynaptic termini, the partitioning of αS between disordered cytosolic and membrane-bound states likely mediates its proposed role in regulation of reserve pools of synaptic vesicles. Previously, we reported on multiple distinct phospholipid binding modes of αS with slow binding kinetics. Here, we report the phospholipid binding properties of the disease variants, viewed by solution NMR in a residue-specific manner. Our results agree qualitatively with previous biophysical studies citing overall decreased lipid affinity for the A30P mutation, comparable affinity for A53T, and an increased level of binding of E46K, relative to wild-type αS. Additionally, our NMR results describe the distribution of lipid-bound states for αS: the population of the SL1 binding mode (residues 3−25 bound as a helix) is augmented by each of the disease variants, relative to wild-type αS. We propose that the SL1 binding mode, which anchors the N-terminus of αS in the lipoprotein complex while the hydrophobic NAC region remains dynamically disordered, is prone to intermolecular interactions which progress toward disease-associated oligomers and fibrils. The elevation of the SL1 binding mode, unchecked by a proportionate population of binding modes incorporating the full N-terminal domain, may well account for the increased toxicity of the A30P, E46K, and A53T disease variants of αS.