Elucidation of Primary (α3N) and Vestigial (α5) Heavy Metal-binding Sites in Staphylococcus aureus pI258 CadC: Evolutionary Implications for Metal Ion Selectivity of ArsR/SmtB Metal Sensor Proteins

Despite a common evolutionary origin, individual members of the ArsR/SmtB family of bacterial metal-responsive transcriptional repressors sense a wide range of heavy-metal ions. The molecular basis for this metal ion selectivity is unclear. Here, we establish that Staphylococcus aureus plasmid pI258 CadC, a Cd(II)/Pb(II)/Bi(III)/Zn(II) sensor, contains two distinct metal-binding sites: a thiolate-rich α3N site comprised exclusively of cysteine ligands that preferentially binds larger, softer metal ions such as Cd(II), Pb(II) and Bi(III); and a second C-terminal α5 site, found at the dimer interface, that is devoid of cysteine ligands and preferentially binds smaller, harder metal ions [Co(II) and Zn(II)] concurrently with metal binding to the α3N site. Optical absorption and X-ray spectroscopies reveal that the α3N site can adopt distinct coordination geometries in order to accommodate different metal ions, i.e. Cd(II), Bi(III), Co(II) and Zn(II) form distorted tetrahedral S4 complexes, while Pb(II) adopts a trigonal S3 complex. Characterization of mutant CadCs reveals that the α3N site is composed of Cys58 and Cys60 from the α3 helix of the helix-turn-helix DNA-binding domain and Cys7 and/or Cys11 from the N-terminal “arm” of CadC; Cys11 is excluded from the Pb(II) coordination sphere. Only the thiolate-rich α3N site is metalloregulatory for repressor binding to a fluorescein-labeled cad O/P oligonucleotide upon coordination to Cd(II), Pb(II), Bi(III), Zn(II), and weakly for Co(II). Substitution of Cys60 and Cys7 with non-ligating residues specifically abrogates metal-dependent negative regulation of cad O/P binding, despite the fact that C60G and C7G CadCs maintain high affinity for metals in altered coordination complexes. These findings reveal that formation of metal coordination bonds to Cys7 and Cys60 play primary roles in transducing the allosteric response in CadC. The evolutionary implications for metal ion selectivity of ArsR/SmtB metal sensor proteins are discussed.