An integrated mass spectrometry and molecular dynamics simulations approach reveals the spatial organization impact of metal-binding sites on the stability of metal-depleted metallothionein-2 species
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Date
2021-10-13
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Mark
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American Chemical Society
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Abstract
Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two alpha- and beta-domains and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. At cellular free Zn(II) concentrations (10(-11)-10(-9) M), MTs do not exist in fully loaded forms with seven Zn(II)-bound ions (Zn7MTs). Instead, MTs exist as partially metal-depleted species (Zn4-6MT) because their Zn(II) binding affinities are on the nano- to picomolar range comparable to the concentrations of cellular Zn(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn(II) (un)binding to MTs, the thermodynamic properties of the Zn1-6MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quantitative bottom-up and topdown MS in combination with steered molecular dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5 mu s) were integrated to unravel the chemical coordination of Zn(II) in all Zn1-6MT2 species and to explain the differences in binding affinities of Zn(II) ions to MTs. Differences are found to be the result of the degree of water participation in MT (un)folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodynamics properties of Zn(II) (un)binding to MT2 are found to differ from those of Cd(II), justifying their distinctive roles. The potential of this integrated strategy in the investigation of numerous unexplored metalloproteins is attested by the results highlighted in the present study.
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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2021, vol. 143, issue 40, p. 16486-16501.
https://pubs.acs.org/doi/10.1021/jacs.1c05495
https://pubs.acs.org/doi/10.1021/jacs.1c05495
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Peer-reviewed
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en