High-Resolution NMR Characterization of the Molecular Interplay between α-Synuclein, Fasudil, and Calcium Ions

Human α-synuclein, an intrinsically disordered protein (IDP) implicated in several neurodegenerative diseases including Parkinson’s disease, consists of 140 amino acids organized into three main regions: a positively charged N-terminal tail, a hydrophobic central region, and a negatively charged C-terminal region. Its dynamic C-terminal region mediates interactions with small molecules and metal ions. Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for characterizing the interactions between IDPs and various binding partners at atomic resolution under physiological conditions.1-4 In this study, we investigate the interaction between α-syn and fasudil, a compound known to bind to the C-terminal part of α-syn and delay the formation of its toxic aggregates.2 We also examine the modulatory role of calcium ions in this interaction. We employed high-resolution NMR spectroscopy, utilizing both 13C and 1H detected experiments and molecular dynamics (MD) simulations, to characterize the interactions between a C-terminal α-syn construct, the small molecule fasudil, and calcium ions. Our study focuses on the side chains of aspartic acid, glutamic acid, and tyrosine residues, which play a key role in the binding of fasudil and calcium ions. We analyse their behaviours in the presence and absence of calcium ions to elucidate the influence of calcium on this process. Side-chain-resolved spectra indicate distinct driving forces for fasudil and calcium ion binding. MD simulations reveal that Ca2+ modifies the local electrostatic environment, decreasing the frequency of fasudil interactions through electrostatic screening and steric effects. Despite this, fasudil retains dynamic, reversible contacts with key tyrosine residues. Overall, the exposed α-synuclein conformations allow for simultaneous, ligand-specific interactions, highlighting side-chain hotspots governing binding in Ca2+rich conditions.