title: Cysteine accessibility during As3+ metalation of the α- and β-domains of recombinant human MT1a.
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.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
authors.forename: Gordon W
authors.initials: GW
authors.lastname: Irvine
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Summers
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stillman
mesh_terms: D000595:Amino Acid Sequence; D001151:Arsenic / Q000737:chemistry*; D016227:Benzoquinones / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D016227:Benzoquinones; C530099:MT1A protein, human; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C004532:quinone; D008668:Metallothionein; D003545:Cysteine; D001151:Arsenic