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.

Vojtech Adam,Artur Krężel,Roman Guran,Vivian De Los Rios,Ondrej Zitka,Manuel David Peris-Díaz,Carmen Domene

doi:10.1021/jacs.1c05495

Abstract

Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two α- and β-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 top-down MS in combination with steered molecular dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5 μs) 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.

Highlights

Mammalian metallothioneins (MTs) are small (∼6−7 kDa) cysteine-rich proteins that participate in the metabolism ofZn(II) and Cu(I)
In order to label free Cys residues, experiments were performed to optimize the IAM concentrations aimed at maintaining the interactions between Zn(II) ions and holoMT2 (Zn7MT2) as well as the 20 Cys residues labeled in the apoMT2 form
Once a quantity was optimized, this amount of IAM was added to the apoMT2 system, which was incubated with 0−7 Zn(II) equiv

Summary

Introduction

Mammalian metallothioneins (MTs) are small (∼6−7 kDa) cysteine-rich proteins that participate in the metabolism ofZn(II) and Cu(I). MTs bind toxic metal ions, such as Cd(II), Pb(II), and Hg(II), limiting their negative effects for cells.[1−5] There are at least a dozen MTs isoforms (MT1−MT4 and their subisoforms) found in the cytosol, nucleus, mitochondria, and the extracellular environment,[1,6,7] which differ in their metal-binding properties and their tissue localization.[1,5,8] MT1 and MT2 are ubiquitously expressed, while MT3 and MT4 are present in the central nervous system and in stratified epithelial tissue, respectively.[1] MTs are 60- to 68-amino acids long and form a dumbbell-shape polypeptide with two thiol-rich regions, separated by a conserved KKS linker containing 11 and 9 cysteinyl residues, designated α- and β-domains.[1−5] The Cterminal α-domain (residues 31−61 in MT2) encloses a Zn4(Cys)[11] cluster formed by five bridging and six terminal sulfur donors. Similar differential binding properties of Zn(II) in human MT3 were postulated.[13,14] This breakthrough revealed that under cellular conditions, where free Zn(II) concentration varies from 10−11 to 10−9 M, MTs exist as partially Zn(II)-depleted species and their speciation depends, inter alia, on the fluctuations of free Zn(II) and apoprotein expression or induction.[15−18] Depending on the fluctuations of Zn(II) concentrations, MTs act as a Received: May 28, 2021 Published: September 3, 2021

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