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). (C) Proposed structure of vesicle-bound αS. (D) Solid-state NMR structure of recombinant αS fibrils (PDB: 2noa). (E) Cryo-EM structure of MSA patient αS fibrils (PDB: 6xyo).

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: αS1-76-MES, 1b: αS1-76-MPAA, 2: αS77-84-Pen77AcK80-NHNH2, 3a: αS1-84-Pen77AcK80-NHNH2. (C) Analytical HPLC trace for the second ligation. 3b: αS1-84_Pen77AcK80-MES, 3c: αS1-84_Pen77AcK80-MTG, 4: αS85-140-C85, 5a: αS-Pen77C85AcK80. (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.

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.

Aggregation kinetics were monitored by fluorescence intensity change of ThT. Time it takes to reach 50% fibrilization (T1/2) 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 = 50 µm. 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.

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