Native mass spectrometry and ultraviolet photodissociation reveal conformation-selectivity of zinc ion to α-synuclein

The conformation-specific binding mechanism between zinc ions （Zn2+） and α-synuclein （α-Syn） plays a pivotal role in elucidating the molecular underpinnings of Parkinson’s disease. α-Syn is an intrinsically disordered protein （IDP）， known for its structural plasticity and dynamic conformational landscape， which are intimately linked to its aggregation propensity and pathogenic potential. In this study， we employed native mass spectrometry （nMS） coupled with 193 nm ultraviolet photodissociation （UVPD） to investigate the α-Syn-Zn2+ interaction across different conformational states. This integrated approach enables both preservation of non-covalent interactions and detailed structural interrogation， offering unparalleled insights into IDP behavior upon metal binding. We identified three distinct charge-state-dependent conformational populations of α-Syn （low-charge， intermediate-charge， and high-charge conformations）， each exhibiting markedly different Zn2+ binding capacities and mechanisms. Experimental data revealed that the low-charge conformations exhibited the highest Zn2+ binding affinity and capacity， accommodating up to three Zn2+ ions. In contrast， the intermediate-charge conformations bound predominantly to one Zn2+ ion. The high-charge conformations， despite their extended structures， retained the ability to bind up to two Zn2+ ions， but with lower affinity. Quantitative analysis of UVPD-derived fragmentation yield changes （ΔFYs） provided residue-level insights into structural perturbations upon Zn2+ coordination. The most pronounced ΔFYs were observed in the low-charge conformers， indicating substantial Zn2+-induced structural stabilization or reorganization， particularly in the C-terminal （Cterm）. Distribution patterns of Zn2+-bound protein fragments （holo fragments） generated by UVPD further supported distinct fragmentation patterns for each conformational state， reflecting differential Zn2+ distribution and protection across the α-Syn sequence. Integrating ΔFYs analysis with holo fragment mapping， we propose three distinct binding mechanisms： （i） low-charge states stabilize Zn2+ binding primarily through electrostatic interactions involving acidic residues in the Cterm； （ii） intermediate-charge states form coordination bonds likely involving histidine or side-chain donors； and （iii） high-charge states exhibit a hybrid mechanism combining electrostatic and coordination elements， though with reduced spatial proximity and structural integrity. Overall， this work highlights the conformation-dependent nature of metal ion interactions in IDPs and underscores the potential of nMS-UVPD as a powerful tool for probing dynamic structural ensembles. These findings provide critical mechanistic insights that could inform the design of conformation-selective therapeutic agents aimed at modulating metal-induced α-Syn aggregation in Parkinson’s disease.