Metal-coordination sphere in the methylated Ada protein-DNA co-complex.
BACKGROUND
The Ada protein of Escherichia coli repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. This residue, Cys69, is ligated to a tightly bound zinc ion in the protein. After methyl transfer, Ada can bind DNA sequence-specifically, inducing the transcription of genes that confer resistance to the toxic effects of methylating agents. Coordination of zinc via a thioether-S is exceedingly rare. We therefore investigated whether methylation causes ligand exchange of Cys69, replacing the thioether with a new zinc ligand with higher affinity for the metal.


RESULTS
We added a 13C-labeled methyl group to Cys69 of Ada and used isotope-edited NMR to observe the behavior of its protons. Comparison of the spectra for the Zn- and 112Cd-bound forms of the methylated protein with that of the 113Cd-bound form provided clear evidence that S-Me-Cys69 is coordinated to the metal in Ada when Ada is bound specifically to DNA.


CONCLUSIONS
The transcriptionally competent form of Ada, in which Cys69 is methylated and the protein is bound to DNA, maintains the coordination of S-Me-Cys69 to the metal ion. Thus, ligand exchange is not responsible for switching Ada from a DNA-repair protein to a transcriptional activator. We propose that the lability of the thioether-zinc coordinate bond may provide a mechanism for down-regulation of the adaptive response by inactivation of the Ada DNA-binding domain.
Myers
L C
LC
Program for Higher Degrees in Biophysics, Harvard University, Cambridge, MA 02138, USA.
Cushing
T D
TD
Wagner
G
G
Verdine
G L
GL
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000737:chemistry*; D029968:Escherichia coli Proteins; D008670:Metals / Q000737:chemistry*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014157:Transcription Factors; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008670:Metals; D014157:Transcription Factors; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D015032:Zinc