α-Synuclein Reduces Tension and Increases Undulations in Simulations of Small Unilamellar Vesicles

Abstract

Using coarse-grained molecular dynamics simulations we have explored the effect of a-Synuclein (aSyn) on the structural and mechanical properties of small unilamellar vesicles in the fluid-phase. The study is motivated by observations that a high density of membrane-bound aSyn inhibits the fusion of synthetic small unilamellar vesicles. By combining three-dimensional pressure tensor calculations with our recently developed spherical harmonics fluctuation analysis approach, we show a reduction in membrane surface tension and increased membrane undulations when aSyn is bound to the vesicle's outer leaflet at a 200:1 L/P. The protein effects these changes by decreasing the negative pressure in the headgroup region of the outer leaflet and increasing the positive pressure throughout the hydrocarbon core.Received for publication 2 December 2014 and in final form 10 March 2015.*Correspondence: jnsachs@umn.edu Multiple in vivo studies have shown that membrane-bound a-Synuclein (aSyn), an amphipathic a-helix that associates with synaptic vesicles (SVs) (1), can disrupt SV trafficking (2). A recent in vitro study showed that aSyn inhibits fusion of small unilamellar vesicles (SUVs) (3), whose size closely matches that of SVs (~30-40 nm diameter). This effect was seen both in DPPC SUVs in the gelphase and, importantly, in a more physiologically relevant quaternary lipid mixture in the fluid-phase (DOPC/DOPE/ SM/Chol). This mixture is highly fusogenic and was originally conceived to closely mimic the lipid composition of synaptic vesicles (4). DeWitt and Rhoades (5) have recently shown that aSyn inhibits SNARE-mediated fusion of fluid-phase SUVs without interacting directly with those proteins.Collectively, these findings have led to the hypothesis that aSyn inhibits fusion through direct alteration of the lipid bilayer's physical properties (2,3,5-7). Once biophysical mechanisms for these inhibitory effects are understood, the impact is expected to extend to other proteins with similar amphipathic characteristics. For example, apolipoprotein A-I, a related, but larger membrane binding protein that shares aSyn's amphipathic 11-mer repeat sequence, was also shown to inhibit vesicle fusion. On the other hand, a short (but still amphipathic) segment of aSyn has no inhibitory effect (3). Exactly what bestows aSyn with its antifusogenic activity, be it sequence-specific or a more generic feature of all amphipathic helices, remains unknown.Because of their small size, SUVs are under high curvature stress, imparting an intrinsic driving force for fusion. In the case of gel-phase SUVs, thermodynamic measurements prompted the hypothesis that aSyn anneals defect zones in the lipid matrix, thus inhibiting SUV fusion by relieving curvature stress (3,7). However, no direct experimental measurements to test this hypothesis have yet been made. Our early molecular dynamics (MD) simulations of aSyn bound to SDS micelles were consistent with this idea, showing that the protein relaxes the highly curved spherical micelle into an ell...

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