Investigation of Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Marie Shimogawa
Ming-Hao Li
Grace Shin Hye Park
Jennifer Ramirez
Hudson Lee
Paris R Watson
Swati Sharma
Zongtao Lin
Chao Peng
Virginia M.-Y Lee
Benjamin A Garcia
David W Christianson
Elizabeth Rhoades
David Eliezer
E James Petersson author has email address

Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, Philadelphia, United States
Department of Biochemistry, Weill Cornell Medicine, New York, United States
Graduate Group in Biochemistry, Biophysics, and Chemical Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States
Department of Biochemistry and Molecular Biophysics, Washington University in St Louis, St Louis, United States
Department of Neurology, David Geffen School of Medicine, University of California - Los Angeles, Los Angeles, United States
Department of Pathology and Laboratory Medicine, Center for Neurodegenerative Disease Research, University of Pennsylvania, Philadelphia, United States
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

https://doi.org/10.7554/eLife.109043.2

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Figures and data

Neurodegeneration-relevant Lys acetylation sites in αS.
(A) αS sequence with positions 12, 21, 23, 32, 43, 45, 58, 60, 80, 96 and 102 marked. (B) Solution NMR structure of micelle-bound αS (PDB: 1qx8) (Ulmer et al., 2005). (C) Proposed structure of vesicle-bound αS based on electron paramagnetic resonance and fluorescence data, where the first ∼100 residues form an amphipathic membrane-bound helix that matches vesicle curvature (Jao et al., 2004; Middleton and Rhoades, 2010). (D) Solid-state NMR structure of recombinant αS fibrils (PDB: 2noa) (Tuttle et al., 2016). (E) Cryo-EM structure of MSA patient αS fibrils (PDB: 6xyo) (Schweighauser et al., 2020).

Semi-synthesis of αS-^AcK₈₀.
(A) Acetylation is introduced through peptide synthesis, and the peptide is combined with expressed peptide fragments using NCL. (B) Analytical HPLC trace for the first ligation. 1a: αS_1-76-MES, 1b: αS_1-76-MPAA, 2: αS_77-84-Pen₇₇^AcK₈₀-NHNH₂, 3a: αS_1-84-Pen₇₇^AcK₈₀-NHNH₂. (C) Analytical HPLC trace for the second ligation. 3b: αS_1-84_Pen₇₇^AcK₈₀-MES, 3c: αS_1-84_Pen₇₇^AcK₈₀-MTG, 4: αS_85-140-C₈₅, 5a: αS-Pen₇₇C₈₅^AcK₈₀. (D) MALDI MS of HPLC-purified αS-^AcK₈₀ (5b).

Expression of αS-^AcK₈₀ through ncAA mutagenesis.
(A) An orthogonal aminoacyl tRNA synthetase (aaRS)/tRNA pair site-specifically incorporates acetyllysine in recombinant αS. Intein tagging at the C-terminus allows for traceless purification of the full-length product. (B) SDS-PAGE gel (Coomassie stain) showing Ni-affinity purification of recombinant αS-^AcK₈₀. Purified αS-^AcK₈₀ (5b) characterized with (C) analytical HPLC and (D) MALDI MS.

Effects of lysine acetylation on micelle-bound αS.
(A) CD spectra for WT and ^AcK₄₃ αS. spectra for other constructs are shown in Appendix 1 – figure 16. (B) Molar ellipticity at 222 nm was normalized to WT value to quantify helicity on SDS micelles. Mean with SD, R=3

Effects of lysine acetylation on in vitro aggregation.
(A) Aggregation kinetics were monitored by fluorescence intensity change of ThT. Two WT traces are shown, one trial conducted with ^AcK₁₂ (squares) and one trial conducted with ^AcK₄₃ and ^AcK₈₀ (circles). ^AcK₁₂ data were scaled by WT trials for clarity. Original data are shown in Appendix 1 – figure 18. (B) Time to reach 50% fibrilization (T_1/2) for each condition was normalized to that of WT. Seeded aggregation was performed with αS monomers where acetylated αS was mixed with αS WT at 25%:75% ratio. SEM, R=6

Effects on aggregation seeding in primary neuron cells.
Left: representative images of neuron cultures treated with unmodified or 25% acetylated αS PFFs, stained with an anti-pS129 antibody (yellow), DAPI (blue), and an anti-MAP2 antbody (red). Scale bar = 25 µm. Larger fields of view shown in Appendix 1 – figure 21. Right: quantification of DAPI-normalized anti-pS₁₂₉ area of intracellular aggregates seeded by different αS PFFs. Mean with SE, R= 11-12. *** = 0.001 < p-value < 0.0001; **** = 0.00001 < p-value < 0.0001

Effects of lysine acetylation on vesicle binding affinity.
(A) NMR intensity ratio for each residue calculated from ¹H-¹⁵N HSQC spectra collected with ¹⁵N-labeled αS variants in the presence or absence of SUVs, normalized by the average ratio for residues 101-140. (B) αS with a TAG codon at the acetylation site of interest and a Cys mutation at a labeling site (8) was co-expressed with an aaRS/tRNA plasmid for acetyllysine incorporation. After intein cleavage, labeling with an Atto488 dye was performed through Cys-maleimide chemistry to give an acetylated, labeled protein (9) for FCS. (C) Vesicle binding affinity determined by fluorescent correlation spectroscopy measurements. For each construct, measurements were performed on three separate days. Mean with SD, R=3

Structural impact of K80 acetylation on fibril morphology.
^AcK₈₀ Fold and WT Fold show the fold of a single αS molecule in the fibrils, viewed down the fibril axis (from ^AcK₈₀-A PBS and WT-A TBS structures). ^AcK₈₀-A and ^AcK₈₀-B show the two fibril polymorphs, with similar protein folds, but different strand-strand packing (from PBS structures). WT-A and WT-B show the two fibril polymorphs, with similar protein folds, but different strand-strand packing (from TBS structures). ^AcK₈₀-A Density Maps show that the same fibril polymorphs were obtained for fibrils made in TBS and PBS. Inset: The interactions of K80 are shown in three previously αS fibril polymorphs designated by their PDB IDs.(Frey et al., 2024; Guerrero-Ferreira et al., 2018; Li et al., 2018b) The overlay shows the similarity of the ^AcK₈₀ fold to the 8pix fold.

Cryo-EM particle numbers for fibril samples

Site-specificity of HDAC activity.
Samples of each of the acetylated αS variants were mixed with HDAC8 and after 24 h, acetylation levels were checked with a MALDI MS assay using ¹⁵N-labeled αS as a standard. Mean with SD, R=3

Semi-synthesis scheme for creating αS-^AcK_80.

Protein semi-synthesis to incorporate ^AcK at position 80.
MALDI-TOF-MS characterization of (A) αS_77-84-Pen₇₇^AcK₈₀-NHNH₂ (2), (B) αS_85-140-C₈₅ (4) and (C) αS_1-84-Pen₇₇^AcK₈₀-MES (3b).

DNA sequences of WT αS expression plasmid.

DNA sequences of WT αS expression plasmid.

ncAA mutagenesis to incorporate acetyllysine at position 12.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 21.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 23.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 32.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 34.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 43.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 45.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 58.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 60.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 96.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

ncAA mutagenesis to incorporate acetyllysine at position 102.
(A) MALDI-MS of purified product (B) SDS-PAGE with Coomassie staining to show affinity purification

Local sequential contexts of amber codon suppression.
(A) Amino acid sequence of αS with acetyl lysine incorporation sites bolded and underlined, (B) Expression yield per liter of E. coli culture for each acetylated construct. Sites 21 and 58 have very similar local sequence context yet gave very different suppression yields.

Individual CD wavelength scans for αS-^AcK constructs.

Individual CD wavelength scans for αS-^AcK constructs.

Aggregation kinetics curves for each αS-^AcK construct.

Aggregation kinetics curves for each αS-^AcK construct.

Effects of 10% αS-^AcK on aggregation kinetics.
The time to reach 50% fibrilization (T_1/2) for each condition was normalized to that of a 100% WT aggregation. Seeded aggregation was performed with αS monomers where acetylated αS was mixed with αS WT at 10%:90% ratio. Mean, with standard error of six replicates.

Primary SDS-PAGE gels for quantifying monomer incorporations of αS-^AcK constructs.
Six replicates shown (R1-6).

Primary SDS-PAGE gels for quantifying monomer incorporations of αS-^AcK constructs.
Six replicates shown (R1-6)

Primary SDS-PAGE gels for quantifying monomer incorporations of αS-^AcK constructs.
Six replicates shown (R1-6).

Primary SDS-PAGE gels for quantifying monomer incorporations of αS-^AcK constructs.
Six replicates shown (R1-6).

Effects of αS-^AcK on total monomer incorporation.
Monomers incorporated into fibrils were quantified by SDS-PAGE gels and normalized to WT values. Mean with standard error, R=6

Neuron imaging data.
Top: Representative images of neuron cultures with additional stains. Yellow = 81A (anti-pS129), Blue = DAPI, Red = MAP2 (Larger fields of view shown than in main text). Bottom: Fluorescence intensity from 81A, DAPI, and MAP2 channels. * = 0.01 < p-value < 0.05; *** = 0.001 < p-value < 0.0001; **** = 0.00001 < p-value < 0.0001

Recombinant ¹⁵N- αS-^AcK₁₂.
(A) MALDI-MS of purified product. (B) SDS-PAGE with Coomassie staining to show affinity purification.

Recombinant ¹⁵N- αS-^AcK₄₃.
(A) MALDI-MS of purified product. (B) SDS-PAGE with Coomassie staining to show affinity purification.

Recombinant ¹⁵N- αS-^AcK₈₀.
(A) MALDI-MS of purified product. (B) SDS-PAGE with Coomassie staining to show affinity purification.

HSQC spectra acquired for αS-^AcK₁₂ in buffer, overlayed with the spectra of αS WT.

HSQC spectra acquired for αS-^AcK₄₃ in buffer, overlayed with the spectra of αS WT.

HSQC spectra acquired for αS-^AcK₈₀ in buffer, overlayed with the spectra of αS WT.

Chemical shift perturbation (Δδ; CSP) from αS-WT calculated at each residue of αS-^AcK₁₂, αS-^AcK₄₃ or αS-^AcK₈₀.

Chemical shift perturbation (Δδ; CSP) calculated at each residue of vesicle-bound αS-WT, αS-^AcK₄₃ or αS-^AcK₈₀.
CSP was calculated by comparing spectra acquired for vesicle-bound state and spectra of free state.

HSQC spectra acquired for free and vesicle-bound αS-WT.

HSQC spectra acquired for free and vesicle-bound αS-^AcK₁₂.

HSQC spectra acquired for free and vesicle-bound αS-^AcK₄₃.

HSQC spectra acquired for free and vesicle-bound αS-^AcK₈₀.

Recombinant αS-C₁₁₄ and fluorescent labeling.
(a) SDS-PAGE with Coomassie staining to show affinity purification. (b) MALDI-MS of purified product αS-C₁₁₄. (c) MALDI-MS of fluorescently labeled, purified product _{αS-C_Atto488114}.

Recombinant αS- ^AcK₁₂C₁₁₄ and fluorescent labeling.
(A) SDS-PAGE with Coomassie staining to show affinity purification. (B) MALDI-MS of purified product αS- ^AcK₁₂C₁₁₄. (C) MALDI-MS of fluorescently labeled, purified product αS- ^AcK₁₂C^Atto488₁₁₄.

Recombinant αS- ^AcK₄₃C₁₁₄ and fluorescent labeling.
(A) SDS-PAGE with Coomassie staining to show affinity purification. (B) MALDI-MS of purified product αS- ^AcK₄₃C₁₁₄. (C) MALDI-MS of fluorescently labeled, purified product αS- ^AcK₄₃C^Atto488₁₁₄.

Recombinant αS- ^AcK₈₀C₁₁₄ and fluorescent labeling.
(A) SDS-PAGE with Coomassie staining to show affinity purification. (B) MALDI-MS of purified product αS- ^AcK₈₀C₁₁₄. (C) MALDI-MS of fluorescently labeled, purified product αS- ^AcK₈₀C^Atto488₁₁₄.

Vesicle FCS data.
FCS autocorrelation curves for αS-C^Atto488₁₁₄ control (WT), αS-^AcK₁₂-C^Atto488_114, αS-^AcK₄₃-C^Atto488₁₁₄, or αS- ^AcK₈₀C^Atto488₁₁₄ with 0.1 mM, 50:50 POPS/POPC vesicles. 30 autocorrelation curves were averaged and fit to a single-component autocorrelation function to determine diffusion time.

Lipid binding affinity of acetylated αS determined by FCS.
Individual binding curves for αS constructs with varying concentrations of 50:50 POPS/POPC vesicles.

TEM images of ^AcK fibrils.
TEM images of fibrils formed from αS monomers comprised of (A-1,A-2,A-3) 25% αS-^AcK₁₂ (B) 25% αS-^AcK₄₃ (C) 25% αS-^AcK₈₀ (D) 100% αS-WT. Multiple fields of view are shown for 25% αS-^AcK₁₂ fibrils to capture the observed heterogeneity in fibril morphology.

Cryo-EM structure from 25% ^AcK₈₀ fibril preparation.
A. Map and model validation. Fourier shell correlations (FSC) curves for density map and model validation. FSC curve for the density map (FSC-masked, blue), refined models versus full maps (FSC-sum, red), half maps for cross-validation (FSC-work, green and FSC-free, purple). Orange solid line and dash line correspond to FSC values of 0.5 and 0.143, respectively. B. Overlay of the cryo-EM map and the atomic model of 25% αS-^AcK₈₀. C. Overlay of 6cu7 (cyan) and 25% αS-^AcK₈₀ (light brown).

Map and model validation.
Fourier shell correlations (FSC) curves for density map and model validation. FSC curve for the density map (FSC-masked, blue), refined models versus full maps (FSC-sum, red), half maps for cross-validation (FSC-work, green and FSC-free, purple). Orange solid line and dash line correspond to FSC values of 0.5 and 0.143, respectively.

Local resolution cryo-EM maps for WT fibrils.
Scale in Å.

Local resolution cryo-EM maps for ^AcK₈₀ fibrils.
Scale bar in Å.

Cryo-EM structures of WT and ^AcK₈₀ fibrils.
Left: Atomic models. Right: Overlays of the cryo-EM maps and the atomic models.

Comparison of cryo-EM density for ^AcK₈₀ fibrils formed in TBS and PBS.

Root mean square deviation (RMSD) of Cα atoms for (left) WT-A and ^AcK₈₀-A and (right) WT-B and ^AcK₈₀-B, calculated using ChimeraX.

Views of cryo-EM density perpendicular to fibril axis for all structures.

Comparison of ^AcK₈₀ fibril structure to other cryo-EM structures.
Overlays performed using alignment tool in PyMOL show similarity to published fibril polymorphs. 8pix/8pic: Frey et. al. (2024) On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation. eLife 12, RP93562. 10.7554/eLife.93562. 6rto/6rtb: Guerrero-Ferreira et al. (2019). Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy. eLife 8, e48907. 10.7554/eLife.48907.

Comparison of ^AcK₈₀ fibril structures to ex vivo cryo-EM structures.
6xyo: Schweighauser et al. (2020). Structures of α-synuclein filaments from multiple system atrophy. Nature. 10.1038/s41586-020-2317-6. Yang, et al. (2022). Structures of α-synuclein filaments from human brains with Lewy pathology. Nature 610, 791-795. 10.1038/s41586-022-05319-3.

MS spectra of ^AcK₁₂ peptide from αS ^AcK₁₂ standard.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation

MS spectra of ^AcK₄₃ peptide from αS ^AcK₄₃ standard.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation.

MS spectra of ^AcK₈₀ peptide from αS ^AcK₈₀ standard.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation.

MS spectra of unmodified K₁₂ peptide from αS WT standard.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation.

MS spectra of unmodified K₄₃ peptide from αS WT standard.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation.

MS spectra of unmodified K₈₀ peptide from αS WT standard.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation.

Representative MS spectra of ^AcK₁₂ peptide from αS in patient sample 2.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation

Representative MS spectra of ^AcK₄₃ peptide from αS in patient sample 15.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation

Representative MS spectra of ^AcK₈₀ peptide from αS in patient sample MSA5-3675.
(A) Extracted ion chromatogram (EIC) (B) MS1 spectrum (C) MS2 spectrum with fragment annotation

Statistics of cryo-EM data collection and refinement for 25% ^AcK₈₀ fibrils

Statistics of cryo-EM data collection and refinement for WT and 100% ^AcK₈₀ fibrils

Quantification of acetylation %ratio by LC-MS/MS data analysis

Ionization Efficiency of Acetylated Tryptic Peptides

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