Cooperativity of α-Synuclein Binding to Lipid Membranes

Abstract

Cooperative binding is a key feature of metabolic pathways, signaling, and transport processes. It provides tight regulation over a narrow concentration interval of a ligand, thus enabling switching to be triggered by small concentration variations. The data presented in this work reveal strong positive cooperativity of α-synuclein binding to phospholipid membranes. Fluorescence cross-correlation spectroscopy, confocal microscopy, and cryo-TEM results show that in excess of vesicles α-synuclein does not distribute randomly but binds only to a fraction of all available vesicles. Furthermore, α-synuclein binding to a supported lipid bilayer observed with total internal reflection fluorescence microscopy displays a much steeper dependence of bound protein on total protein concentration than expected for independent binding. The same phenomenon was observed in the case of α-synuclein binding to unilamellar vesicles of sizes in the nm and μm range as well as to flat supported lipid bilayers, ruling out that nonuniform binding of the protein is governed by differences in membrane curvature. Positive cooperativity of α-synuclein binding to lipid membranes means that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. The phenomenon described in this work may have implications for α-synuclein function in synaptic transmission and other membrane remodeling events.

Keywords: Adair equation; Cooperative binding; fluorescence correlation spectroscopy; homotropic allostery; lipid membrane; α-synuclein.

Figure 1

α-Synuclein binding to GUVs. (A, B) Two examples of bright field (left panels) and fluorescence (middle and right panels) images of samples containing DOPC:DOPS 7:3 GUVs and α-synuclein-647, with the protein below the saturation concentration, corresponding to the excess of membrane surface. In the right panels, GUVs missing from the fluorescence images are indicated with yellow dashed circles. (C) Size comparison of a GUV of 5 μm diameter and unfolded α-synuclein (approximated to a dot of 5 nm in radius) showing that for α-synuclein, the membrane of a GUV appears completely flat. (D) Cartoon showing the distribution of protein molecules (red) in a population of vesicles (blue) for the cases of independent binding (left) and fully cooperative binding (right).

Figure 2

Binding of α-synuclein-647 to DOPC:DOPS 7:3 SUVs with 0.5% Oregon Green DHPE (SUV diameter ≈70 nm). Results from the fluorescence correlation spectroscopy experiment: (A) total number of vesicles N_ves (blue circles) and the number of vesicles having protein bound N_ves+αsyn (red circles) extracted from the background-corrected amplitudes of the 488 and 633 nm autocorrelation curves, respectively. (B) Theoretical predictions of the number of vesicles having protein bound as a function of L/P for the cases of independent (purple dashed line) and infinitely cooperative binding (gray dotted line). The solid blue line corresponds to the total number of vesicles. In the calculations the protein concentration was kept constant while the lipid concentration was varied. Details of the calculations are presented in Experimental Section. (C) Brightness per vesicle in the red channel as a function of L/P. (D) Total red fluorescence signal from vesicles (i.e., with the α-synuclein signal subtracted) as a function of L/P. The autocorrelation and cross-correlation curves for free α-synuclein and α-synuclein with SUVs at L/P of 50, 200, and 2000 are presented in Figure S5.

Figure 3

α-Synuclein binding to a flat supported lipid bilayer. (A) Fluorescent signal from a 7:3 POPS:DOPS SLB incubated for 15 min with (top) 0.1 nM α-synuclein and (bottom) 10 nM α-synuclein. (B) (Top) Single molecule fluorescence image of 12 pM α-synuclein adsorbed on a bare glass slide. The scale bar is 20 μm for all images. (Bottom) Histogram showing the total intensity per detected fluorescence ”spot” in the single molecule fluorescence image. The dashed red line is a Gaussian fit to the main peak, showing that 90% of the detected spots exhibit intensity of less than 20 units, corresponding to a monomeric form of the protein (n = 335). (C) Density of α-synuclein bound to a SLB for bulk concentrations in the range 0.1–500 nM determined from the fluorescence signal. All data points are the mean ± SE from two or three separate measurements, and the inset shows the first six data points with expanded y axis. The dotted blue line shows a fit of the Adair equation (eq 4) for one binding site and corresponds to independent binding. The red, purple, and gray lines represent fits of the Adair equation with two, three, and four coupled binding sites, respectively.

Figure 4

α-Synuclein binding to SUVs studied with cryo-TEM. (A–C) Examples of cryo-TEM images of small unilamellar vesicles (SUVs) with α-synuclein at different lipid/protein ratios. The images of samples at L/P of 50, 100, 200, and 1500 are presented in Figure S7. The scale bar is the same as in (C) for all images. (D) Percentage of deformed vesicles (mean ± SD) calculated from six different images of each sample from one experiment assuming no fusion of vesicles. The number of analyzed vesicles was 5330.

Figure 5

Calculations using the Adair equation: fractions of hypothetical vesicles with n proteins bound as a function of total protein concentration for vesicles with 2 (upper panels) and 10 binding sites (lower panels) for the cases of no cooperativity (left) and strong positive cooperativity (right). Values of the macroscopic binding constants for the case of independent binding were calculated from K_j = × 10⁶ M^–1 where N is the total number of binding sites. Values of the macroscopic binding constants for positively cooperative binding to vesicles with two binding sites were K₁ = × 10⁶ M^–1 and K₂ = 4 × 10⁶ M^–1, assuming an average affinity of 1 × 10⁶ M^-1 and a free energy coupling of ΔΔG=-10 kJ mol^-1. For the case of vesicles with 10 binding sites and cooperative binding, the macroscopic binding constants were K₁ = 0.0745 M^-1, K₂ = 2.15 M^-1, K₃ = 81.4 M^-1, K₄ = 3400 M^-1, K₅ = 1.5 × 10⁵ M^-1, K₆ = 6.7 × 10⁶ M^-1, K₇ = 2.9 × 10⁸ M^-1, K₈ = 1.2 × 10¹⁰ M^-1, K₉ = 4.7 × 10¹¹ M^-1 and K₁₀ = 1.3 × 10¹³ M^-1, assuming an average affinity of 1 × 10⁶ M^-1 and a free energy coupling of ΔΔG=-10 kJ mol^-1.