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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 (Zn_4-6MT) 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 Zn_1-6MT2 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 Zn_1-6MT2 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.

SUBMITTER: Peris-Diaz MD

PROVIDER: S-EPMC8517974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:What are the molecular determinants of protein-protein binding affinity and whether they are similar to those regulating fold stability are two major questions of molecular biology, whose answers bring important implications both from a theoretical and applicative point of view. Here, we analyze chemical and physical features on a large dataset of protein-protein complexes with reliable experimental binding affinity data and compare them with a set of monomeric proteins for which melting temperature data was available. In particular, we probed the spatial organization of protein (1) intramolecular and intermolecular interaction energies among residues, (2) amino acidic composition, and (3) their hydropathy features. Analyzing the interaction energies, we found that strong Coulombic interactions are preferentially associated with a high protein thermal stability, while strong intermolecular van der Waals energies correlate with stronger protein-protein binding affinity. Statistical analysis of amino acids abundances, exposed to the molecular surface and/or in interaction with the molecular partner, confirmed that hydrophobic residues present on the protein surfaces are preferentially located in the binding regions, while charged residues behave oppositely. Leveraging on the important role of van der Waals interface interactions in binding affinity, we focused on the molecular surfaces in the binding regions and evaluated their shape complementarity, decomposing the molecular patches in the 2D Zernike basis. For the first time, we quantified the correlation between local shape complementarity and binding affinity via the Zernike formalism. In addition, considering the solvent interactions via the residue hydropathy, we found that the hydrophobicity of the binding regions dictates their shape complementary as much as the correlation between van der Waals energy and binding affinity. In turn, these relationships pave the way to the fast and accurate prediction and design of optimal binding regions as the 2D Zernike formalism allows a rapid and superposition-free comparison between possible binding surfaces.

Dataset Information

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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