α-Synuclein's Uniquely Long Amphipathic Helix Enhances its Membrane Binding and Remodeling Capacity

Abstract

α-Synuclein is the primary protein found in Lewy bodies, the protein and lipid aggregates associated with Parkinson's disease and Lewy body dementia. The protein folds into a uniquely long amphipathic α-helix (AH) when bound to a membrane, and at high enough concentrations, it induces large-scale remodeling of membranes (tubulation and vesiculation). By engineering a less hydrophobic variant of α-Synuclein, we previously showed that the energy associated with binding of α-Synuclein's AH correlates with the extent of membrane remodeling (Braun et al. in J Am Chem Soc 136:9962-9972, 2014). In this study, we combine fluorescence correlation spectroscopy, electron microscopy, and vesicle clearance assays with coarse-grained molecular dynamics simulations to test the impact of decreasing the length of the amphipathic helix on membrane binding energy and tubulation. We show that truncation of α-Synuclein's AH length by approximately 15% reduces both its membrane binding affinity (by fivefold) and membrane remodeling capacity (by nearly 50% on per mole of bound protein basis). Results from simulations correlate well with the experiments and lend support to the idea that at high protein density there is a stabilization of individual, protein-induced membrane curvature fields. The extent to which these curvature fields are stabilized, a function of binding energy, dictates the extent of tubulation. Somewhat surprisingly, we find that this stabilization does not correlate directly with the geometric distribution of the proteins on the membrane surface.

Keywords: Alpha-Synuclein; Membrane remodeling; Tubulation.

Figure 1

Circular dichroism spectra for αSyn₇₈ (red) and αSyn₁₀₀ (blue) free in solution (solid lines) and incubated with POPG vesicles (dashed lines). Both αSyn₇₈ and αSyn₁₀₀ fold into an α-helical conformation when bound to POPG vesicles.

Figure 2

Representative EM images of tubules formed by αSyn₁₀₀ (A) and αSyn₇₈ (B). Scale bar is 200 nm. Red circles highlight vesicles in the αSyn₇₈ system.

Figure 3

(A) FCS traces for αSyn₇₈ and αSyn₁₀₀ in the absence (black or magenta) or presence of POPG (red or blue). (B) Vesicle clearance absorbance traces for POPG + buffer (black), POPG + αSyn₇₈ at equal protein concentration (red), POPG + αSyn₇₈ at equal mass of protein (orange), and POPG + αSyn₁₀₀ (blue). (C) Tubulation capacities for αSyn constructs relative to buffer.

Figure 4

Time-averaged height surfaces, h(x,y), for αSyn₇₈ (left) and αSyn₁₀₀ (right) for low-density (A) and high-density (B) systems. Purple star indicates N-terminus of protein. The white arrows highlight local region of on-average positive curvature suggesting an alignment of proximal proteins. (C) Excess area per protein for αSyn₇₈ (left) and αSyn₁₀₀ (right) systems [low-density, blue; high-density (equal-mass, brown; equal-protein, green)]. (D) Protein angle distributions for αSyn₇₈ and αSyn₁₀₀ high-density systems (αSyn₁₀₀, grey-bars; αSyn_{78,eq-protein}, black-stem; αSyn_78,eq-mass, red-curve).

Figure 5

Heptad depth profile for αSyn constructs. Depth profiles for αSyn₇₈ (blue), αSyn₉₃ (green), and αSyn₁₀₀ (red) are parsed by heptad and illustrate differential penetration relative to the POPG headgroup (black dashed-line).