High-Field NMR Insights into Side-Chain Mediated Interactions of α-Synuclein with Small Molecules

Intrinsically disordered proteins (IDPs), such as α-synuclein, play central roles in numerous physiological and pathological processes, yet their structural characterization at atomic resolution remains predominantly restricted to the polypeptide backbone. Amino acid side chains, such as aromatic, polar, charged and methyl groups, are critical drivers that modulate molecular recognition and interaction specificity; however, their exploration is severely hindered by the extensive resonance overlap arising from high structural flexibility and disorder. To address this limitation, we show how optimized experiments acquired at high magnetic fields allow us to resolve resonances of side chains in IDPs to investigate the binding mechanisms of α-synuclein with small-molecule ligands. Our approach demonstrates that while long-range electrostatic interactions primarily dictate the initial targeting of small molecules to specific regions of the disordered sequence, the binding events are locally modulated and stabilized by specific side-chain groups. Remarkably, these interactions occur dynamically, fully preserving the structurally disordered nature of the protein matrix. This work underlines the importance of expanding the observable NMR parameter space to side chains to decode the subtle details of the driving forces governing IDP-ligand complexes. This information lays the basis for innovative approaches to target IDPs.