title: Ratiometric pulsed alkylation mass spectrometry as a probe of thiolate reactivity in different metalloderivatives of Staphylococcus aureus pI258 CadC.
abstract: The coordination structure and reactivities of metal ligands in metal-sensing metalloregulatory coordination complexes may well dictate their biological properties. Here, we use the technique of ratiometric pulsed alkylation mass spectrometry (rPA-MS) to probe the structure and reactivities of metal coordination complexes formed by different metalloderivatives of Staphylococcus aureus plasmid pI258-encoded CadC, the metal-regulated transcriptional repressor of the cad operon. The cad operon provides resistance to large thiophilic heavy metal pollutants including Cd(II), Pb(II), and Bi(III). Two cysteines, an invariant Cys7 and a conserved Cys11, separated by three amino acids near the N-terminus of each subunit within dimeric CadC, donate two of the four coordination bonds to Cd(II) and Bi(III); in contrast, Cys11, but not Cys7, is excluded from the trigonal Pb(II) complex. rPA-MS reveals that Cys7 is strongly protected from alkylation in all metal complexes, Pb(II) being most effective, reducing k(app)(C7)by approximately 1000-fold relative to apo-CadC; in contrast, the reactivity of Cys11 is indistinguishable from that of apo-CadC, consistent with an S(3) coordination complex. Only in the tetrathiolate complexes formed by Cd(II) and Bi(III) is the reactivity of Cys11 appreciably reduced, but only by >or=10-fold. These data suggest that the Cys11-S(-)-metal coordination bond or that side of the coordination chelate in the trigonal Pb(II) complex defines a "weak point" in the chelate and thus might provide an entry site for potential metal ligand exchange reactions important for metal resistance in vivo. In contrast, Cys7 forms a tight coordination bond with all inducing metals, consistent with its role as a critical allosteric ligand in the metalloregulation of the operator/promoter binding.
authors.affiliation: Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Julius L
authors.initials: JL
authors.lastname: Apuy
authors.forename: Laura S
authors.initials: LS
authors.lastname: Busenlehner
authors.forename: David H
authors.initials: DH
authors.lastname: Russell
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
mesh_terms: D000478:Alkylation; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001729:Bismuth / Q000737:chemistry; D002104:Cadmium / Q000737:chemistry; D002918:Chymotrypsin / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D005033:Ethylmaleimide / Q000737:chemistry; D006868:Hydrolysis; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; C080050:CadC protein, Bacteria; D008667:Metalloproteins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D002104:Cadmium; D003035:Cobalt; D002918:Chymotrypsin; D014357:Trypsin; D015032:Zinc; D003545:Cysteine; D005033:Ethylmaleimide; D001729:Bismuth