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Comparative Study
. 2010 Feb 9;49(5):862-71.
doi: 10.1021/bi901723p.

Differential phospholipid binding of alpha-synuclein variants implicated in Parkinson's disease revealed by solution NMR spectroscopy

Christina R Bodner  1 , Alexander S Maltsev, Christopher M Dobson, Ad Bax
Affiliations
Free PMC article
Comparative Study

Differential phospholipid binding of alpha-synuclein variants implicated in Parkinson's disease revealed by solution NMR spectroscopy

Christina R Bodner et al. Biochemistry. .
Free PMC article

Abstract

Three familial variants of the presynaptic protein alpha-synuclein (alphaS), A30P, E46K, and A53T, correlate with rare inherited Parkinson's disease (PD), while wild-type alphaS is implicated in sporadic PD. The classic manifestation of both familiar and sporadic PD is the formation of fibrillar structures of alphaS which accumulate as the main component in intraneuronal Lewy bodies. At presynaptic termini, the partitioning of alphaS between disordered cytosolic and membrane-bound states likely mediates its proposed role in regulation of reserve pools of synaptic vesicles. Previously, we reported on multiple distinct phospholipid binding modes of alphaS with slow binding kinetics. Here, we report the phospholipid binding properties of the disease variants, viewed by solution NMR in a residue-specific manner. Our results agree qualitatively with previous biophysical studies citing overall decreased lipid affinity for the A30P mutation, comparable affinity for A53T, and an increased level of binding of E46K, relative to wild-type alphaS. Additionally, our NMR results describe the distribution of lipid-bound states for alphaS: the population of the SL1 binding mode (residues 3-25 bound as a helix) is augmented by each of the disease variants, relative to wild-type alphaS. We propose that the SL1 binding mode, which anchors the N-terminus of alphaS in the lipoprotein complex while the hydrophobic NAC region remains dynamically disordered, is prone to intermolecular interactions which progress toward disease-associated oligomers and fibrils. The elevation of the SL1 binding mode, unchecked by a proportionate population of binding modes incorporating the full N-terminal domain, may well account for the increased toxicity of the A30P, E46K, and A53T disease variants of alphaS.

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Figures

Figure 1
Figure 1
Signal attenuation observed in the 1H−15N HSQC spectrum of αS corresponds to the fractional population of αS engaged in phospholipid binding at a particular residue location. (A) Cross section of the 1H−15N HSQC spectrum of E46K αS, in the absence (black) and presence (red) of 0.03% SUVs. (B) Fractional signal attenuation of wild-type (black), A30P (blue), E46K (gold), and A53T (red) αS in the presence of 0.03% (w/v) SUVs plotted as function of residue number. The protein concentration for all series is 300 μM, such that the lipid to protein molar ratio is approximately 4:3.
Figure 2
Figure 2
Effect of increased lipid concentrations on the binding of αS and its disease variants. Fractional intensity in the HSQC is shown for WT (black), A30P (blue), E46K (gold), and A53T (red) αS in the presence of 0.25 (A) or 2.0% SUVs (B). The lipid:protein ratio is 11:1 or 86:1, respectively. N-Terminal residues whose intensities are too weak for reliable detection have been omitted. For C-terminal residues, which show slightly heterogeneous chemical shifts in the 2.0% SUV condition (see the text), fractional intensities plotted in panel B reflect the attenuation of the nonshifted resonance.
Figure 3
Figure 3
Transverse relaxation rates of the 15N TROSY component, R2T, measured for 600 μM WT αS (white circles) or A30P αS (gray circles) in the presence of 0.06% SUVs. The rates measured for WT αS in the absence of lipids are also plotted (black circles).
Figure 4
Figure 4
Series of strip plots showing 1H−1H amide NOEs for residues 25−38 (excepting P30) of 600 μM A30P αS in the presence of 0.06% SUVs. Strips are taken orthogonal to the 1H frequency axis of a 600 MHz 3D HMQC-NOESY-HMQC spectrum. The NOE mixing time was 100 ms.
Figure 5
Figure 5
Series of strip plots showing 1H−1H amide NOEs for residues 25−38 of 600 μM E46K αS in the presence of 0.06% SUVs. Strips are taken orthogonal to the 1H frequency axis of a 600 MHz 3D HMQC-NOESY-HMQC spectrum. The NOE mixing time was 100 ms.
Figure 6
Figure 6
Schematic depicting the proposed increased vulnerability of αS to self-association when engaged in the SL1 binding mode vs SL2. The A30P and A53T disease mutations both increase the relative population of SL1, for which a long unstructured stretch of N-terminal residues (including the hydrophobic NAC region) is exposed.
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