Unravelling the mechanistic details of metal binding to mammalian metallothioneins from stoichiometric, kinetic, and binding affinity data

Abstract

Metallothioneins (MTs) are small, cysteine-rich proteins, found throughout Nature. Their ability to bind a number of different metals with a range of stoichiometric ratios means that this protein family is critically important for essential metal (Zn and Cu) homeostasis, metal storage, metal donation to nascent metalloenzymes as well as heavy metal detoxification. With its 20 cysteines, metallothionein is also considered to protect cells against oxidative stress. MT has been studied by a large number of researchers over the last 6 decades using a variety of spectroscopic techniques. The lack of distinguishing chromophores for the multitude of binding sites has made the evaluation of stoichiometric properties for different metals challenging. Initially, only Cd-NMR spectroscopy could provide strong evidence for the proposed cluster formation of Cd-MT. The extraordinary development of electrospray ionization mass spectrometry (ESI-MS), where all coexisting species in solution are observed, revolutionized MT research. Prior to the use of ESI-MS data, a range of "magic numbers" representing metal-to-MT molar ratios were reported from optical spectroscopic studies. The availability of ESI mass spectral data led to (i) the confirmation of cluster formation, (ii) a conceptual understanding of the cooperativity involved in multiple metal binding events, (iii) the presence of domain specificity between regions of the protein and (iv) mechanistic details involving both binding affinities and rate constants. The kinetic experiments identified the presence of multiple individual binding sites, each with a unique rate constant and an analogous binding affinity. The almost linear trend in rate constants as a function of bound As provided a unique insight that became a critical step in the complete understanding of the mechanistic details of the metalation of MT. To fully define the biological function of this sulfur-rich protein it is necessary to determine kinetic rate constants and binding affinities for the essential metals. Recently, Zn competition experiments between both of the isolated fragments (α and β) and the full-length protein (βα-MT 1a) as well as Zn competition between βα-MT 1a and carbonic anhydrase were reported. From these data, the trend in binding affinities and the values of the K of the 7 bimolecular reactions involved in metalation were determined. From the analysis of ESI-MS data for Cu binding to βα-MT 1a at different pH-values, a trend in the 20 binding affinities for the complete metalation mechanism was reported. This review details a personal view of the historical development of the determination of stoichiometry for metal binding, the structure of the binding sites, the rates of the metalation reactions and the underlying binding affinities for each metalation step. We have attempted to summarize the experimental developments that led to the publication in May 2017 of the experimental determination of the 20 binding constants for the 20 sequential bimolecular reactions for Cu binding to the 20 Cy...

ZnT2 is an electroneutral proton-coupled vesicular antiporter displaying an apparent stoichiometry of two protons per zinc ion

“…As a consequence, the energies presented are not adjusted to the effective zinc concentration, and the absolute value of the energy can be misleading. We proceeded with the assumption that the effective zinc concentration is fairly high in the cytoplasm (where zinc-carrying metallothioneins are abundant [37,38]), which would result in a decrease in energy for the zinc bound states at the cytoplasm.…”

Arsenic Species in Cordyceps sinensis and Its Potential Health Risks

“…These proteins including hemoglobin, metallothionein, thioredoxin peroxidase, glutathione reductase, and glycopeptide peroxidase (Mizumura et al, 2010; Shen et al, 2013; Chen et al, 2015; Liu Q et al, 2018). Metallothionein, a metal-binding protein having cysteine residues and high metal content, was founded in living organisms (Ziller et al, 2017; Scheller et al, 2018). Therefore, we speculate that uAs could specifically bind to proteins in C. sinensis .…”

Metallothioneins of the urochordateOikopleura dioicahave Cys-rich tandem repeats, large size and cadmium-binding preference

“…4,5 From a structural perspective, MTs have been extensively analyzed showing that they are able to coordinate a number of heavy metal ions through the formation of metal-thiolate bonds. 6,7 Depending on their metal-binding preferences, MTs can be classified in a stepwise gradation from extreme Zn/Cd-thioneins to extreme Cu-thioneins. 8,9 The metal-binding preference of a given MT cannot be, however, directly predicted from its amino acid sequence, and metal-binding assays have to be performed to classify newly discovered forms.…”