Binding adaptability of chemical ligands to polymorphic α-synuclein amyloid fibrils.

α-synuclein (α-syn) assembles into structurally distinct fibril polymorphs seen in different synucleinopathies, such as Parkinson's disease and multiple system atrophy. Targeting these unique fibril structures using chemical ligands holds diagnostic significance for different disease subtypes. However, the molecular mechanisms governing small molecules interacting with different fibril polymorphs remain unclear. Here, we investigated the interactions of small molecules belonging to four distinct scaffolds, with different α-syn fibril polymorphs. Using cryo-electron microscopy, we determined the structures of these molecules when bound to the fibrils formed by E46K mutant α-syn and compared them to those bound with wild-type α-syn fibrils. Notably, we observed that these ligands exhibit remarkable binding adaptability, as they engage distinct binding sites across different fibril polymorphs. While the molecular scaffold primarily steered the binding locations and geometries on specific sites, the conjugated functional groups further refined this adaptable binding by fine-tuning the geometries and binding sites. Overall, our finding elucidates the adaptability of small molecules binding to different fibril structures, which sheds light on the diagnostic tracer and drug developments tailored to specific pathological fibril polymorphs.

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

α-Synuclein forms amyloid fibrils that are critical in the progression of Parkinson's disease and serves as the pathological hallmark of this condition. Different posttranslational modifications have been identified at multiple sites of α-synuclein, influencing its conformation, aggregation and function. Here, we investigate how disease-related phosphorylation and O-GlcNAcylation at the same α-synuclein site (S87) affect fibril structure and neuropathology. Using semi-synthesis, we obtained homogenous α-synuclein monomer with site-specific phosphorylation (pS87) and O-GlcNAcylation (gS87) at S87, respectively. Cryo-EM revealed that pS87 and gS87 α-synuclein form two 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. Phosphorylation at the same site prevents a lengthy C-terminal region including residues 73 to 140 from incorporating into the fibril core due to electrostatic repulsion. Instead, the N-terminal half of the fibril (1-72) takes on an arch-like fibril structure. We further show that both pS87 and gS87 α-synuclein fibrils display reduced neurotoxicity and propagation activity compared with unmodified α-synuclein fibrils. Our findings demonstrate that different posttranslational modifications at the same site can produce distinct fibril structures, which emphasizes link between posttranslational modifications and amyloid fibril formation and pathology.