FIGURE 5.

Poly (ADP-ribose) interacts with αSyn via electrostatic forces in the N-terminal region of the protein. (A) Alignment of the full αSyn sequence with PBMs yielded a 59% PAR-binding probability at amino acid residues 43–54 and 48–58 on αSyn. Lysine amino acid residues (red) at the two sites were substituted to neutral alanine residues. (B) Three mutants of αSyn were generated with compromised PAR-binding sites. Two of the αSyn mutants had a point mutation at amino acid residues K43 and K58, respectively, while the third mutant had two point mutations at positions K43 and K45. All mutants were fully fibrillated within 72 h. Scale bar 200 nm. (C) PAR immunodot blot (top panel), whereby WT and mutant αSyn fibrils were spotted onto a membrane, along with, histone H4 (positive control), and BSA (negative control) and incubated with PAR polymer to assess PAR binding. Semi-quantitative analysis (bottom panel) of WT and mutant αSyn fibril signal intensity normalized to WT αSyn signal. One-way ANOVA (n = 3). ****P < 0.0001. (D) Cryo-EM structure of MSA Type I αSyn fibril interacting with the PAR-dimer complex with a low free binding energy of –15.6 kcal/mol.