Phosphorylation and O-GlcNAcylation at the same α-synuclein site generate distinct fibril structures

ABSTRACT α-Synuclein (α-syn) forms amyloid fibrils that are critical in the progression of Parkinson’s disease (PD) and serves as the pathological hallmark of PD. Different posttranslational modifications (PTMs) have been identified at multiple sites of α-syn, influencing its conformation, aggregation and function. Here, we investigate how disease-related phosphorylation and O-GlcNAcylation at the same α-syn site (S87) affect fibril structure and neuropathology. Using semi-synthesis, we obtained homogenous α-syn monomer with site-specific phosphorylation (pS87) and O-GlcNAcylation (gS87) at S87, respectively. Cryo-EM analysis revealed that pS87 and gS87 α-syn form two novel but distinct fibril structures. The GlcNAc situated at S87 establishes interactions with K80 and E61, inducing a unique iron-like fold with the GlcNAc molecule on the iron handle. While, phosphorylation at the same site prevents a lengthy C-terminal region including residues 73-140 from incorporating into the fibril core due to electrostatic repulsion. Instead, the N-terminal half (1-72) shapes a novel arch-like fibril structure. We further show that both pS87 and gS87 α-syn fibrils display reduced neurotoxicity and propagation activity compared with unmodified α-syn fibril. Our findings demonstrate that different PTMs at the same site can produce distinct fibril structures, which emphasizes the precise regulation of PTMs to amyloid fibril formation and pathology.