Complementary Structural and Chemical Biology Methods Reveal the Basis for Selective Radioligand Binding to...

Fibrillar aggregation of α-synuclein (αSyn) is a hallmark of Parkinson's disease (PD) and related disorders, including multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Despite advances in αSyn fibril structural characterization, the relevance of in vitro and ex vivo structures to patient aggregates remains unclear, particularly for developing therapeutic or diagnostic molecules. Cryo-electron microscopy (cryo-EM) studies of αSyn fibrils with ligands often reveal binding at multiple sites, likely due to high ligand concentrations. Here, various structural and chemical biology techniques were used to characterize αSyn fibrils in the presence of EX-6, a candidate ligand for positron emission tomography (PET) imaging of synucleinopathies. Transmission electron microscopy (TEM) and cryo-EM revealed no significant fibril core changes upon binding. Förster resonance energy transfer (FRET) further demonstrated that the disordered C-terminus was unaltered. Cryo-EM and crosslinking mass spectrometry (XL-MS) identified consistent binding sites, with one (Site 2*) providing a well-defined pocket for high-resolution analysis. Site 2* showed similar residue positioning in MSA patient-derived structures, suggesting MSA selectivity. [ ³ H]-EX-6 binding assays demonstrated a 10-fold preference for MSA over PD tissue, with autoradiography further confirming MSA selectivity. Taken together, the combined use of structural and chemical biology techniques provides a comprehensive understanding of EX-6 binding that would not be possible with any single method. Optimization of ligand-protein and ligand-ligand interactions observed in the cryo-EM structure will enable the development of EX-6 as a PET imaging probe for MSA.