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

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.

The Bioinorganic Chemistry of Mammalian Metallothioneins

“…158,215 Very recently a mass spectrometric investigation supported by molecular dynamics calculations characterized the coordination environments in all seven metalated species of human MT2 and demonstrated how the coordination changes upon successive metal association/dissociation. 216 Remarkably, the binding of Zn(II) is different from that of Cd(II). Thus, Zn(II)-and Cd(II)-MTs are isostructural only in the species with seven metal ions, limiting the use of Cd(II) for investigations of MTs in zinc metabolism.…”

“…Very recently a mass spectrometric investigation supported by molecular dynamics calculations characterized the coordination environments in all seven metalated species of human MT2 and demonstrated how the coordination changes upon successive metal association/dissociation . Remarkably, the binding of Zn­(II) is different from that of Cd­(II).…”

“…Order of the stepwise Zn­(II) dissociation for the unfolding pathway for (A) Zn 7 MT2 and (B) Cd 7 MT2 obtained by constant-speed steered molecular dynamics simulations that involve Zn 7 MT2 and Cd 7 MT2 with the C-termini fixed . The bars show the binding/unbinding of the metal ion for each metal-loaded species.…”

“…Zn­(II) and Cd­(II) are represented by gray spheres, and the sulfur atoms are shown in yellow . The figure is adopted from ref with permission of the American Chemical Society.…”

Exploration of high dimensional free energy landscapes by a combination of temperature‐accelerated sliced sampling and parallel biasing

“…This problem was addressed to some extent by selectively grouping CVs 51 . The method has been applied to a wide spectrum of complex chemical and biological problems; See references 52–60; see also references 39, 61. Several other attempts to improve the sampling using a MTD‐like bias have also been proposed lately 25,49,62–64 …”