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 ( approximately 10(12)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 alpha4 helix (Cys58/60). We show here that metal ion binding to this site (denoted the alpha3N 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 ( approximately 10(5)-10(7)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+/-0.4s(-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 alpha3N 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.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Laura S
LS
Busenlehner
David P
DP
Giedroc
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C080050:CadC protein, Bacteria; D008670:Metals; D012097:Repressor Proteins