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Biochemical and biophysical research communications

Cysteine accessibility during As3+ metalation of the α- and β-domains of recombinant human MT1a.


PMID 23523794

Abstract

Metallothionein is a ubiquitous metal binding protein that plays an important role in metal ion homeostasis and redox chemistry within cells. Mammalian metallothioneins bind a wide variety of metals including the metalloid As3+ in two domains (β and α) connected by a short linker sequence. Three As3+ bind in each domain for a total of 6 As3+ per protein. In recombinant human metallothionein (rh-MT1a) each As3+ binds three cysteine residues to form As3Cys9(CysSH)2-α-rhMT1a in the 11 Cys α-domain and As3Cys9-β-rhMT1a in the 9 Cys β-domain. This means that there should be 2 free cysteines in the α-domain but no free cysteines in the β-domain. By using benzoquinone, the number and relative accessibility of the free cysteinyl thiols during the metalation reactions were determined. The electrospray ionization mass spectrometry (ESI-MS) data confirmed that each As3+ binds using exactly 3 cysteine thiols and showed that there was a significant difference in the reactivity of the free cysteines during the metalation reaction. After a reaction with two molar equivalents of As3+ to form As2Cys6(CysSH)3-αβ-rhMT1a, the remaining 3 Cys in the 9 Cys β-domain were far less reactive than those in the α-domain. Molecular dynamics calculations for the metalation reactions with As3+ measured by ESI-MS allowed an interpretation of the mass spectral data in terms of the relative location of the cysteine thiols that were not involved in As3+ coordination. Together, these data provide insight into the selection of a specific cysteinyl thiol by the incoming metals during the stepwise metalation of metallothioneins.