Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

Abstract

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.

19F NMR studies of α‐synuclein‐membrane interactions

“…13,20,24,28,31,32 Solution NMR is a powerful tool for accessing processes that occur over a range of timescales. 26 In 250 mM NaCl, SDS forms larger rod-like micelles, decreasing the tumbling rate of the a-synuclein-micelle complex. The slow tumbling results in the absence of a detectable resonance for residue 39 [ Fig.…”

“…Although these studies indicate that the properties of phospholipid membranes (i.e., charge, curvature, size, and the identity of the acyl chains) affect binding, there is no consensus, and some studies are contradictory. 21,22,[24][25][26][27] We show that 19 F NMR is another useful technique for assessing a-synuclein-membrane interactions. 19 F is a good reporter of conformational changes due to its sensitivity to the environment and the fact that few natural biological molecules contain fluorine.…”

“…This supposition, however, is inconsistent with our data, which agree with the conclusion of Bodner et al that the first 25 residues adopt a helical structure state which anchors the interaction to SUVs. 26,27 In that model, a-synuclein prefers SUV defects, which are affected by head group size, charge, and the hydrophobic thickness. The reduced binding of a-synuclein to DMPC/DMPG SUVs is due to the increase in the curvature that arises as a consequence of the acyl chain, which agree with the conclusion of Nuscher et al 45 As we observe, these properties affect a-synuclein-membrane interactions.…”

“…These data show that the Nterminal region interacts strongly with POPG SUVs while the C-terminal region is involved in weaker membrane interactions, a conclusion consistent with other 15 N NMR studies. 26,27 Conclusions Binding mainly involves the N-terminal region of a-synuclein, and both the head group and the acyl chain of phospholipids are important. Although the protein prefers negatively charged lipids, other properties, including the saturation of the lipid also affect a-synuclein membrane interactions.…”

“…More specifically, 15 N intensity and relaxation data from titration of SUVs sugget there exists several binding modes in which the first 25 residues adopt a helical state that anchor the interaction with SUVs. 26,27 A variety of other techniques, including circular dichroism spectropolarimetry, 12,13,19,28 fluorescence spectroscopy, 21,[29][30][31] and EPR [21][22][23][24][25] have been used to study how the phospholipid composition of vesicles affects a-synuclein affinity. Although these studies indicate that the properties of phospholipid membranes (i.e., charge, curvature, size, and the identity of the acyl chains) affect binding, there is no consensus, and some studies are contradictory.…”