Multiple tight phospholipid-binding modes of alpha-synuclein revealed by solution NMR spectroscopy

Abstract

'In dopaminergic neurons, alpha-synuclein (alphaS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of alphaS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which alphaS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of alphaS. Exchange between the free state and the lipid-bound alphaS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s(-1). Partitioning of the binding modes is dependent on lipid/alphaS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of alphaS adopts an alpha-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound alphaS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large alpha-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this "dark state." Titration of alphaS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where alphaS lies in an alpha-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by alphaS.

Fig. 1

Effects of addition of lipid SUVs on the ¹H-¹⁵N HSQC spectrum of αS, recorded at a ¹H frequency of 600 MHz, 293 K, pH 6.0. (a) A section from the assigned and overlaid HSQC spectra of 150 μM αS in the presence of 0.03% (black) or 0.06% SUV (red). (b) Fractional signal attenuation relative to lipid-free spectra as a function of residue number for 150 μM αS in the presence of 0.03% SUV (w/v). The lipid: αS molar ratio is 2.6:1. Missing values correspond to proline residues (marked by arrows), two solvent-exchanged N-terminal residues, and residues whose ¹H/¹⁵N cross peaks are significantly overlapped, which have been eliminated from further analysis.

Fig. 2

Impact of αS-lipid binding on ¹H-¹⁵N HSQC cross peak intensity and line width under lipid-limited and lipid-saturated conditions. (a) In the presence of 0.03% SUV, NMR cross peak signal attenuation profiles for decreasing concentrations of αS are shown: 150 μM (blue), 75 μM (green), 25 μM (red), and 12.5 μM (black). (b) Attenuation profile for 150 μM αS in the presence of 2.0% SUV, where the lipid: αS ratio is 173:1. Cross peak intensities of residues preceding V52 were too weak for reliable measurements. Decreased αS concentration or increased lipid availability reduces competition between different binding modes, altering the attenuation profile. Upon lowering the αS concentration, no significant changes to the profile are observed for lipid: αS ratios > 15, where binding appears no longer constrained by the lipid-limited condition. (c) ¹H (filled circles) and ¹⁵N (open circles) line widths for the same 25 μM αS sample used for data in panel (a). Narrow and uniform line widths show that observed signals correspond to amide groups of residues in the random coil state.

Fig. 3

Fluorescence anisotropy, r, of 500 μM αS as a function of lipid concentration. The emission maximum of tyrosine, 306 nm, is monitored. At the lipid concentration of 0.1%, where the lipid: αS ratio is 2.6:1, the change in fluorescence anisotropy nears saturation.

Fig. 4

Transverse relaxation rates of the ¹⁵N TROSY component (R₂^T) measured for 600 μM αS in the presence (black) and absence (red) of 0.03% SUV, measured at 600 MHz ¹H frequency. In the presence of lipids, the effective R₂^T is the sum of the intrinsic R₂^T and the forward exchange rate, k_on. Rates are derived from the mono-exponential fitting of intensities recorded in an interleaved manner for twelve transverse decay times: 20, 30, 50, 70, 90, 120, 150, 180, 220, 270, 400, and 500 ms.

Fig. 5

Lipid to αS NOE magnetization transfer as monitored by fractional attenuation of the ¹H-¹⁵N HSQC intensities, when the HSQC spectrum is preceded by selective saturation of phospholipid methylene resonance at 1.16 ppm. The sample contains 150 μM perdeuterated αS and 0.03% SUV. The spectra with and without lipid saturation are recorded in an interleaved mode, applying a 1.5 s presaturation with a 21 Hz RF field strength at either 1.16 ppm or at −10.0 ppm . The strongest NOE transfer is observed for N-terminal residues which also exhibit the highest signal attenuation, indicative of highest partitioning into the “dark state”. Error bars correspond to the estimated uncertainty calculated from the signal-to-noise ratio of each correlation.

Fig. 6

Series of strip plots for residues 77-90, taken orthogonal to the ¹H frequency axis of a 600-MHz 3D HMQC-NOESY-HMQC spectrum labeled with ¹⁵N frequencies in both F₁ and F₂ dimensions, recorded for 600 μM αS in the presence of 0.03% SUV and using a 100 ms NOE mixing time. The use of two ¹⁵N dimensions in this experiment capitalizes on the greater dispersion of ¹⁵N frequencies relative to ¹H frequencies to resolve ambiguities in the poorly dispersed spectrum of αS. Spin diffusion undergone by αS in the helical bound state manifests itself in the extensive i to i ± n amide cross peaks.

Fig. 7

NOE effect from Leu-C^δ1 H₃ methyl groups to backbone amide protons, as monitored by the intensity of ¹⁵N-¹H HSQC correlations for a sample of 600 μM (I,L,V)- αS in the presence of 0.06% SUV, when the ¹⁵N-¹H HSQC spectrum is preceded by the selective inversion or non-inversion of the Leu C^δ1 methyl protons (see Methods), using a 250 ms NOE mixing period. The protein is uniformly enriched in ¹⁵N and ²H, with selectively-labeled ¹³C¹H₃ groups for Ile, Leu, and Val. (a) Section of the difference HSQC spectrum showing differential intensities of leucine and their sequentially adjacent residues. (b) Corresponding histogram plotting the fractional change in signal intensity as a function of protein sequence; positions of leucine residues are marked by arrows.

Fig. 8

Cryo EM images for a sample of 0.03% DOPE:DOPS:DOPC SUVs in the absence (a) and presence (b-d) of 600 μM αS. In the absence of αS, vesicles are relatively uniform, 20–40 nm in diameter. Destabilization of the vesicle by αS interaction results in gross rearrangements, with the formation of large, multilamellar (b), tubular (c), and pinched, branched, and tubular structures (d). The dark ridges in the top left of (a) and (d) and bottom left of (c) correspond to the boundaries of the carbon support of the grid.

Fig. 9

Pulsed field gradient diffusion plots for αS and SUVs, isolated and in mixed samples. 150 μM αS in absence of lipid (open circles), and in the presence of 0.03% (grey circles) or 2.0% (black circles) lipid. 2.0% DOPE:DOPS:DOPC SUV in the absence (open squares) of αS. The plot for free αS shows a linear correlation, while all other series show differing degrees of non-linearity attributable to the heterogeneity of the species being measured. Diffusion delays of 500ms were used and for αS the signal decay of the entire amide envelope region is used for intensity measurement. For the 150 μM αS/0.03% SUV sample, 68% of the observed total intensity corresponds to lipid-free protein (note that for lipid-bound protein, only about half of the residues yield observable intensity). Plotted is the natural logarithm of the ratio between the observed total amide proton intensity, I, and the value I_o measured at 6% of maximum gradient strength (87 G/cm), i.e., G_o = 5.2 G/cm. In order to avoid contamination from direct solvent exchange with amide protons, data were recorded at pH 6.0, 10 °C.