Zn2+ Binding Shifts the Conformational Ensemble of α-Synuclein Monomers toward Accelerated Amyloid Formation.

Alpha-synuclein (αS) is an intrinsically disordered protein (IDP) that can self-assemble into amyloid fibrils, undergoing a transition from disordered monomers to ordered β-sheet-rich fibrils. The amyloid state of αS is implicated in various synucleinopathies, most notably Parkinson's disease (PD), in which αS fibrils accumulate as insoluble Lewy body deposits. Colocalized with αS in Lewy bodies are elevated levels of metal ions including Zn²⁺. We find <i>in vitro</i> that Zn²⁺ accelerates aggregation of N-terminally acetylated αS, decreasing the <i>t</i>₅₀ ca. 5-fold, as measured by thioflavin T (ThT) fluorescence. Strikingly, the extent of Zn²⁺ binding (native mass spectrometry; MS) and shifts of the monomeric αS conformational ensemble toward compaction, measured using ion mobility MS (IM-MS) at different αS:Zn²⁺ ratios, mirror precisely the accelerated aggregation kinetics. Chemical shift perturbations in Nuclear Magnetic Resonance (NMR) spectroscopy were investigated together with molecular dynamics (MD) to map the Zn²⁺ binding sites and subsequent effects on conformation under identical solution conditions to those used in IM-MS. Zn²⁺ is found to predominantly interact with negative residues in the C-terminal region of αS but also His50 in the N-terminal region. This promiscuity in interactions potentially guides compaction of the protein chain by bridging residues between the N- and C-terminal regions through Zn²⁺ ion co-ordination. This study provides insights into the early stages of amyloid assembly, correlating aggregation kinetics with structural compaction in monomeric αS and highlighting the capability of native IM-MS to resolve complex structural ensembles of a disordered protein.