Selectivity of methylation of metal-bound cysteinates and irs consequences

Alkylation of metal-bound cysteinate residues forms an integral step in bot h the activation of the DNA-damage sensing Ada protein from E. coli and the reaction mechanisms of several zinc-dependent enzymes. The roles of metal ions and the protein structure in regulating the reactivity of bound cystei nate residues is not well-understood. Variants of a consensus zinc finger p eptide were used to determine the effects of alkylation of cysteine residue s on both metal binding and stability of the peptide structure. The ability of thioethers to act as ligands was probed through the direct synthesis of peptides with methionine or S-methylcysteine replacing the second histidin e within the zinc finger framework. This position can be substituted with c ysteine with no significant loss of structure or stability. Two-dimensional H-1 NMR studies and water exchange experiments of the peptide with S-methy lcysteine in this position showed that methylation affected the structure o f the peptide-zinc complex in the last turn of the helix, adjacent to the s ite of methylation, without disrupting the rest of the structure. Titration s with cobalt revealed that the peptides with methionine or S-methylcystein e do not bind metal ions as tightly as do peptides with histidine or cystei ne in this position. Similar to peptides. lacking a fourth ligand, these th ioether containing peptides form two-to-one peptide-to-cobalt complexes at low metal concentrations. Alkylation of the cobalt complex of the peptide w ith cysteine as the fourth ligand with dimethyl sulfate in aqueous solution yielded a product with absorption spectral features essentially identical with those of the S-methylcysteine derivative. Methylation of either of the other two cysteine residues within this peptide resulted in the loss of de tectable metal binding. The carboxyl terminal cysteine was alkylated at a r ate approximately 5-fold higher than the other cysteine residues, potential ly due to the relative accessibility. of this cysteine sulfur compared with the others which are shielded by peptide amide to sulfur hydrogen bonds. O ther studies suggest that all of the cysteine residues in this peptide are less prone to alkylation in the cobalt complex than they are in the unfolde d, metal-free form under similar solution conditions. These results indicat e that thioether residues have a significantly lower affinity for cobalt(II ) and zinc(II) than cysteine or histidine. Thus, significant modulation of metal-bound cysteinate reactivity can be achieved through the position of t he cysteinate within the three-dimensional structure of a metal-peptide com plex.