Interaction of Human α-Synuclein and Parkinson's Disease Variants with Phospholipids

Abstract

␣-Synuclein has been centrally implicated in neurodegenerative disease, and a normal function in developmental synaptic plasticity has been suggested by studies in songbirds. A variety of observations suggest the protein partitions between membrane and cytosol, a behavior apparently conferred by a conserved structural similarity to the exchangeable apolipoproteins. Here we show that the capacity to bind lipids is broadly distributed across exons 3, 4, and 5 (encoding residues 1-102). Binding to phosphatidylserine-containing vesicles requires the presence of all three exons, while binding to phosphatidic acid can be mediated by any one of the three. Consistent with a "class A2" helical binding mechanism, lipid association is disrupted by introduction of charged residues along the hydrophobic face of the predicted ␣-helix and also by biotinylation of conserved lysines (which line the interfacial region). Circular dichroism spectroscopy reveals a general correlation between the amount of lipid-induced ␣-helix content and the degree of binding to PS-containing vesicles. Two point mutations associated with Parkinson's disease have little (A30P) or no (A53T) effect on lipid binding or ␣-helicity. These results are consistent with the hypothesis that ␣-synuclein's normal functions depend on an ability to undergo a large conformational change in the presence of specific phospholipids.

19F NMR studies of α‐synuclein‐membrane interactions

“…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.…”

“…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. [32][33][34][35] Additionally, adding a few fluorine atoms to a protein has a minimal effect on structure and dynamics.…”

“…[8][9][10][11][12] The protein binds lipids and anionic detergents through the seven imperfect, cationic, 11-amino acid repeats located in its N-terminal and hydrophobic regions. [6][7][8][13][14][15][16][17][18][19][20] Electron paramagnetic resonance (EPR) data on its complex with small unilamellar vesicles (SUVs) suggest that the first $100 residues of the monomeric protein adopt an a-helical conformation that lies on the membrane surface. [21][22][23][24][25] The last $40 residues lack defined structure and do not appear to be involved in 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.…”

“…32,33,35,36 Furthermore, the methods listed above provide only the overall affinity of a-synuclein for membranes. 19 F NMR detects a-synuclein-membrane interactions at the level of individual residues. Here, we use 19 F NMR as a probe to monitor systematically the binding of a-synuclein to SUVs with different head groups and acyl chains.…”