Metallothioneins (MTs) are cysteine- and metal-rich proteins that play central roles in Cu(I)/Zn(II) homeostasis and detoxification. In mammals, four MT isoforms exist (MT-1/4). Human MTs are monomeric proteins of 61-68 amino acids characterized by a conserved array of 20 cysteines all involved in coordination of d10 metal ions in two separated metal-thiolates clusters. Despite their sequence and metal coordination similarity, the MT-3 isoform possess a unique copper-thionein character that results in structural and biological functions not shared by other isoforms. MT-3 shows neuronal growth inhibitory activity, and protects neurons from the toxic effect of amyloid-β (Aβ). Our work demonstrated how the MT-3 reactivity towards Cu(II) controls aberrant copper-protein interactions and amyloid aggregation. MT-3 scavenges Cu(II) from soluble, oligomeric, and aggregated amyloidogenic proteins and peptides via metal-swap reactions. Zn7MT-3 thiolates efficiently reduce Cu(II) to Cu(I) and bind it to form Cu(I)4Zn4MT-3 via cooperative formation of a unique redox-inert Cu(I)4-thiolate cluster in its β-domain, in which metal-metal interactions exists. This reactivity underlies its protective effect against amyloid-Cu(II) toxicity. We have addressed the structural features underlying the unique copper-thionein character in MT-3 over other MTs. We here define the process of Cu(II) reduction and Cu(I)-thiolate cluster assembly in MT-3, its Cu(I) binding and redox-silencing properties, and compare them to other MT isoforms. By using spectroscopic and spectrometric techniques we reveal a novel sequential pathway for Cu(I)4-thiolate cluster formation in which unique long-lived metal-stabilized thiyl and disulphide radical anions are formed. By conducting mutational studies on MT-3 in comparison to MT-2, which possess a zinc-thionein character, we revealed how non-coordinating residues are instrumental for outer-sphere control of Cu(I) selectivity bias in MT-3 metal-thiolate clusters. The study contributes to the fundamental understanding on how sequence, structure and metal coordination overall determine metal selectivity, reactivity and function in MTs.

Principles of metal selectivity bias in metallothionein metal-thiolate clusters

256th National Meeting (2018)

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