The soft metal ion binding sites in the Staphylococcus aureus pI258 CadC Cd(II)Pb(II)/Zn(II)-responsive repressor are formed between subunits of the homodimer

The Staphylococcus aureus plasmid pI258 CadC is a homodimeric repressor that binds Cd(II), Pb(II), and Zn(II) and regulates expression of the cadAC operon. CadC binds two Cd(II) ions per dimer, with a tetrathiolate binding site composed of residues Cys⁷, Cys¹¹, Cys⁵⁸, and Cys⁶⁰. It is not known whether each site consists of residues from a single monomer or from residues contributed by both subunits. To examine whether Cys⁷ and Cys¹¹ are spatially proximate to Cys⁵⁸ and Cys⁶⁰ of the same subunit or of the other subunit, homodimers with the same cysteine mutation in each subunit and heterodimers containing different cysteine mutations in the two subunits were reacted with 4,6-bis(bromomethyl)-3,7- dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione, which cross-links thiol groups that are within 3-6 A of each other. Cys⁷ or Cys¹¹ cross-linked only with Cys⁵⁸ or Cys⁶⁰ on the other subunit. The data demonstrate that Cys⁷ and Cys¹¹ from one monomer are within 3-6 Å of either Cys⁵⁸ or in the other monomer. The results of this study strongly indicate that each of the two Cd(II) binding sites in the CadC homodimer is composed of Cys⁷ and Cys¹¹ from one monomer and Cys⁵⁸ and Cys⁶⁰ from the other monomer.