Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties

Abstract

N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson's disease, is common in mammals. The impact of this modification on the protein's structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone (3)J(HH) couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and (3)J(HH) data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an "initiation-elongation" process of binding to the membrane.

Figure 1

Impact of N-terminal acetylation on the NMR spectrum of aS. (A) Overlay of a small expanded region of the 800 MHz HSQC spectra of nonacetylated (black) and acetylated (red) aS. The pronounced change in the peak position for Ser9 is marked with an arrow. For the overlay of full spectra, see Figure S1 of the Supporting Information. Differences in chemical shifts in parts per million between acetylated and nonacetylated aS for different backbone atoms as a function of residue number: (B) ¹³C^α, (C) ¹³C^β, (D) ¹³C′, (E) ¹⁵N, and (F) ¹H^N. Pairwise rmsds calculated over residues 13–140 are 0.008 ppm (¹³C^α), 0.007 ppm (¹³C^β), 0.005 ppm (¹³C′), 0.019 ppm (¹⁵N), and 0.002 ppm (¹H^N).

Figure 2

Population of α-helix for nonacetylated (black) and acetylated (red) WT aS as derived from the measured ¹³C^α, ¹³C^β, ¹³C′, ¹⁵N, and ¹H^N chemical shifts using δ2D.

Figure 3

CD data for the interaction of nonacetylated (black) and acetylated (red) WT aS with lipid vesicles. (A) Solid lines represent CD spectra obtained at a 75:1 lipid:protein molar ratio. Dashed lines show CD spectra in the absence of lipids. (B) Graph showing the change in CD signature at 222 nm, reflecting the amount of α-helical structure, as a function of lipid:protein molar ratio. Buffer conditions were 20 mM phosphate (pH 6) and 150 mM NaCl. Measurements were performed on samples containing 10 μM protein. The lipid consisted of 30% DOPS, 50% DOPE, and 20% DOPC.

Figure 4

Linearized (logarithmic) PFG diffusion plots for nonacetylated (black) and acetylated (red) WT aS. Proton peak intensities were observed in the methyl region. Dashed lines represent linear fits to the symbols of the matching color.

Figure 5

Ratios of intraresidue to sequential H^α–H^N NOE intensities, d_αN(i,i)/d_αN(i–1,i), for the first 10 residues of the nonacetylated (black) and acetylated (red) 15-residue N-terminal peptides. The ratio for residue G7 was divided by 2, and the ratio for L8 was multiplied by 2 to roughly account for the presence of two H^α protons on G7. Ratios for the full-length, nonacetylated protein can be found in ref (36).

Figure 6

Ratios of WT aS TROSY-HSQC peak heights in the presence (I) and absence (I_o) of lipids. Data from the following three samples are presented: acetylated aS at a lipid:protein (L:P) ratio of 22 (red circles), nonacetylated aS at an L:P ratio of 22 (black triangles), and nonacetylated aS at an L:P ratio of 44 (black circles). Experiments were performed on 200 μM protein samples in 20 mM phosphate (pH 6) and 150 mM NaCl buffer at 15 °C on an 800 MHz spectrometer. The lipid consisted of 15% DOPS, 25% DOPE, and 60% DOPC.

Figure 7

Overlay of small expanded regions of the TROSY-HSQC spectra of N-terminally acetylated, C-terminally His-tagged aS after soft purification (red) and of acetylated WT aS that had undergone heating during purification (black), recorded under matching buffer conditions [1× PBS, 5% glycerol, 0.05% BOG, and 7% D₂O (pH 6)]. Spectra were acquired on a 500 MHz spectrometer equipped with a cryoprobe. The signature position of the S9 peak indicates that both proteins are indeed N-terminally acetylated. The only peaks that show small chemical shift differences between the two samples are in the acidic C-terminal region (e.g., S129) and result from the direct effect of the His tag. The full spectrum is shown in Figure S6 of the Supporting Information.