In dopaminergic neurons, a-synuclein (aS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of aS to membrane phospholipids is implicated in its functional role of synaptic regulation, but also impacts fibril formation associated with Parkinsons disease. We describe here a solution NMR study in which aS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles;the results provide evidence for multiple distinct phospholipid-binding modes of aS. Exchange between the free and lipid-bound aS 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:aS 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 N-terminus of aSadopts an a-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 aS 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 a-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this dark state. Titration of aS 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 aS lies in an a-helical conformation on the membrane surface, and instead suggest that considerable remodeling of the vesicles is induced by aS.
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