Mammalian metallothioneins (MTs) are a group of small ((~ 6-7 kDa) cysteine-rich proteins that bind various metal ions including essential Zn(II) and Cu(I), and toxic ones such Cd(II) or Hg(II). There are at least a dozen mammalian MTs that are classified into subfamilies (MT1-MT4), which differ in their metal-binding properties and tissue localization. Mammalian MTs are 60-68 amino acid long, and render a dumbbell-shape polypeptide with two thiol-rich domains. The N-terminal folds into the β-domain enclosing a Zn4(Cys(S))11 and the C-terminal forms the α-domain including a Zn3(Cys(S))9 cluster. A decade ago, using sensitive zinc fluorescent probes, it was demonstrated that Zn(II) ions bind to MT2 with different affinities varying from nanomolar to picomolar range. Therefore, at cellular free Zn(II) concentrations (10-11-10-9 M), MTs do not exist in fully loaded forms with seven Zn(II) ions (Zn7MTs) but as partially metal-depleted species (Zn4-6MT). At present, commonly established methodologies lack enough spatial resolution to characterise metal-depleted states and MT structures still remain elusive. Here, we integrated native mass spectrometry (MS), label-free quantification bottom-up and top-down MS and molecular dynamics (MD) simulations to probe (i) the mechanostability, (ii) the spatial organization of Zn(II) ions in partially loaded states, and (iii) the coordination number and other structural properties that render Zn1-6MT stable species. The combination of MS and MD employed here has addressed these questions with the highest resolution to date and provides a unique frame of reference to study how speculative ZN-depleted states function. The potential of this integrated strategy in the challenging investigation of stability-structure-function relationships of numerous unexplored metalloproteins is attested by the structural accounts highlighted in the present study.

Spatial organization and stability of metallothionein-2 species involved in cellular zinc buffering

ACS Fall 2021

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