Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement

Abstract

␣-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, ␣-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of ␣-synuclein bound to a lipid bilayer. Continuous-wave EPR was used to assign local secondary structure and to determine the membrane immersion depth of lipid-exposed residues, whereas pulsed EPR was used to map long-range distances. The structure of ␣-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that ␣-synuclein forms an extended, curved ␣-helical structure that is over 90 aa in length. The monomeric helix has a superhelical twist similar to that of right-handed coiled-coils which, like ␣-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd ‫؍‬ 0.82 Å). The ␣-synuclein helix extends parallel to the curved membrane in a manner that allows conserved Lys and Glu residues to interact with the zwitterionic headgroups, while uncharged residues penetrate into the acyl chain region. This structural arrangement is significantly different from that of ␣-synuclein in the presence of the commonly used membranemimetic detergent SDS, which induces the formation of two antiparallel helices. Our structural analysis emphasizes the importance of studying membrane protein structure in a bilayer environment.EPR ͉ Parkinson's disease ͉ fibril-forming proteins ͉ 11-aa repeats T he interaction of ␣-synuclein with membranes is thought to be important in its physiologic function in vivo, as well as in its misfolding and aggregation in the pathogenesis of Parkinson disease (1-10). Although the function of ␣-synuclein in vivo is not fully understood, it has been observed to localize to presynaptic nerve termini, where it modulates presynaptic pool size and neurotransmitter release (11-16). These functions are likely to be mediated by the interaction of ␣-synuclein with synaptic vesicles, and in vitro studies have shown that ␣-synuclein interacts strongly with highly curved vesicles that are similar in size to synaptic vesicles (17, 18). The structural characterization of membrane-bound ␣-synuclein is significant, given the importance of membrane interactions to the pathologic and physiologic roles of ␣-synuclein.Previous studies have revealed that the interaction of monomeric ␣-synuclein with negatively charged vesicles induces a predominantly ␣-helical structure located in the N-terminal region of the protein (17,19,20). This region contains seven 11-aa-repeat regions that share some sequence similarity with apolipoproteins [supporting information (SI) Fig. S1]. Sequence analysis using algorithms for apolipoproteins predicts the formation of five separate helices (17). However, no high-resolutio...

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.…”

“…In the a-synuclein-membrane interaction model, [21][22][23][24][25]31 $100 N-terminal residues lie on the membrane surface as an extended helix, and the remaining residues are disordered. Accordingly, the buried acyl chain should have little effect on a-synuclein interactions.…”

“…SUVs were prepared as described. 25 Briefly, the lipids were weighed to produce the desired ratios and dissolved in CHCl 3 . The solvent was evaporated by using a gentle stream of N 2 (g).…”

“…[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. 13,21 Recent solution NMR data, however, appear incompatible with this model.…”

“…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.…”