Kinetics of metal binding by the toxic metal-sensing transcriptional repressor Staphylococcus aureus pI258 CadC

The mechanisms by which metal ions are sensed in bacterial cells by metal-responsive transcriptional regulators (metal sensor proteins) may be strongly influenced by the kinetics of association and dissociation of specific metal ions with specific metalloregulatory targets. Staphylococcus aureus pI258-encoded CadC senses toxic metal pollutants such as Cd(II), Pb(II) and Bi(III) with very high thermodynamic affinities (≈1012 M−1) in forming either distorted tetrahedral (Cd/Bi) or trigonal (Pb) coordination complexes with cysteine thiolate ligands derived from the N-terminal domain (Cys7/11) and a pair of Cys in the α4 helix (Cys58/60). We show here that metal ion binding to this site (denoted the α3N or type 1 metal site) is characterized by two distinct kinetic phases, a fast bimolecular encounter phase and a slower intramolecular conformational transition. Metal association rates are fast (≈105–107 M−1 s−1) and strongly dependent on the metal ion type in a manner that correlates with metal specificity in vivo. In contrast, the observed rate of the slower isomerization step is independent of the metal ion type (2.8 ± 0.4 s−1) but is reduced 6-fold upon substitution of Cys7, a key metal ligand that drives allosteric negative regulation of DNA binding. Chelator (EDTA)-mediated metal dissociation rates from the α3N site are extremely slow (⩽10−4 s−1). Where observable dissociation can be observed, a ternary CadC–metal ion-chelator complex is invoked, suggesting that metal–ligand exchange may be an important factor in metal sensing and resistance in the cell.