Metalation and Demetalation of Human Metallothionein Studied by Ion Mobility Mass Spectrometry

Abstract

The mechanism of cadmium binding to intact human metallothionein-2A (MT) is investigated. We describe two complementary mass spectrometry (MS) strategies to study the metalation/demetalation mechanism: (i) chemical labeling combined with proteomics-based top-down or bottom-up approaches (ii) ion mobility-mass spectrometry (IM-MS). We presented here for the first time for direct identification of metal-binding sites on a partially-metalated intermediates by combing chemical modifications (N-ethylmaleimide (NEM)) and 2D MS-CID-IM-MS. These results provide conclusive evidence of specific binding of Cd^2+ to the α-domain and demetalation is the reverse order of metalation. Molecular-specific profiles for metal affinity and kinetic stability for each metalated intermediate provided by MS reveal that the reactivity of various CdiMT species is dependent on their metalation state and reaction is cooperative for both domains. These data suggest that the α-domain has greater thermodynamic/kinetic stability than does the β-domain. In addition, the alkylation on kinetically labile cysteins reveals that the α domain contains a weakly coordinated site at Cys33, which is located at the extreme N terminus of the α domain and might be related to the intra-domain cooperativity.

Further, IM-MS and molecular dynamics (MD) simulations are used to follow metal-induced conformational changes, which reveal new insights into the mechanism for metalation of MT. IM data for the CdiMT (i = 0 − 7) ions reveal a diverse population of ion conformations. Upon metal-ion binding, the conformational diversity for apoMT and partially-metalated ions converges toward ordered, compact conformations as the number of bound Cd^2+ ions increase. We interpret this data as evidence of metal-dependent folding and increasing numbers of metal ions yield more compact ion structures. MD simulations provide additional information on conformation candidates of CdiMT (i = 0 − 7) that supports the convergence of distinct conformational populations upon metal binding. Integrating the IM-MS and MD data provides a global view that shows stepwise conformational transition of an ensemble as a function of metal ion bound. Furthermore, collisional activation was used to increase effective ion temperature (Teff) prior to entering and within the ion mobility analyzer, providing new insights about protein folding energy landscape.