Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.

PMCID: PMC3591419This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils

Structural Intermediates during α-Synuclein Fibrillogenesis on Phospholipid Vesicles

α-Synuclein (AS) fibrils are the main protein component of Lewy bodies, the pathological hallmark of Parkinson’s disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to β-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to β-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the <i>N</i>-terminal domain with a partial disruption of the long β-strand located in the 40s and small perturbations in residues located in the “non-β amyloid component” (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles

Fibrils of mutant AS proteins prepared <i>in vitro</i> have a highly homogeneity and morphology similar to WT fibrils.

<p>(A) AS fibrils formation of (blue circles) E46K, (red triangles) A53T and (black squares) WT monitored by the Thioflavin T fluorescence assay. Error bars were determined from seven replicates for each. Measurements were normalized to the highest fluorescence intensity obtained across all samples. (B) Comparison of the electron micrographs of (top) E46K, (middle) A53T and (bottom) WT AS fibrils. ¹³C-¹³C 2D with 50 ms DARR mixing of (C) E46K and (D) A53T AS fibrils.</p

Sequential backbone-walk used to obtain the chemical shift assignments of A53T.

<p>Illustration of backbone connectivity through the NCACX (red), NCOCX (blue) and CAN(co)CX (black) spectra of residues A90-E83. In all cases the homonuclear mixing was achieved with 50 ms DARR.</p

The A53T mutation causes minor perturbations throughout the AS fibril sequence.

<p>(A) Expansions of ¹³C-¹³C 2D spectral overlays (50 ms DARR mixing, 600 MHz ¹H frequency and 13.3 kHz MAS) of WT (black) and A53T (red) AS fibril samples. (B) Plot of the chemical shift perturbations between WT and A53T chemical shifts versus residue number. Residues labeled as (*) correspond to perturbations above 1 ppm. Residues labeled as (#) correspond to glycines. The mutation is indicated with (†). Error bars correspond to the chemical shift variations from one WT batch to another. WT chemical shift assignments were obtained from the BMRB #16939.</p

The E46K mutation causes major chemical shift perturbations throughout the AS fibril sequence.

<p>(A) Expansions of ¹³C-¹³C 2D spectral overlays (50 ms DARR mixing) of WT (black) and E46K (blue) AS fibril samples. (B) Plot of chemical shift perturbations between WT and E46K chemical shifts versus residue number. Residues labeled as (*) correspond to perturbations greater than 5 ppm (¹⁵N) or 3 ppm (¹³C). Residues labeled as (#) correspond to glycines. The mutation is indicated with (†). Error bars correspond to the chemical shift variations from one WT batch to another. WT chemical shift assignments were obtained from the BMRB #16939.</p

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution

TALOS+ predicted backbone dihedral angles ψ and φ as a function of residue number.

<p>(A) E46K and (B) A53T AS fibrils. Error bars based on the 10 best TALOS+ database matches. Representation of the secondary structure for WT (black), E46K (blue) and A53T (red) AS fibrils based on TALOS+ analysis (β-strands, arrows; turn or loop curved lines; not predicted, dashed lines). WT TALOS+ results based on those from Comellas <i>et al</i>.</p