Chemical communication across the zinc tetrathiolate cluster in Escherichia coli Ada, a metalloactivated DNA repair protein.
The Escherichia coli Ada protein repairs methylphosphotriesters in DNA through direct, irreversible transfer to a cysteine residue on the protein, Cys 69. Methylation of Cys 69 increases the sequence-specific DNA-binding activity of Ada by 10(3)-fold, enabling the methylated protein to activate transcription of a methylation-resistance regulon. The thiolate sulfur atom of Cys 69 is coordinated to a tightly bound zinc ion in the Ada N-terminal domain, and this metal-ligand interaction plays a direct role in promoting the DNA repair chemistry. Ada is thus the founding member of a mechanistic class of proteins that employ metalloactivated thiolates as nucleophiles, other examples of which include protein prenyltransferases and cobalamin-independent methionine synthase. Here we have probed the role of the three other Cys residues in Ada that together with Cys 69 coordinate the zinc through mutation to the alternative ligand residues Asp and His. All of the mutant proteins folded properly and bound zinc, but none of them exhibited measurable levels of DNA repair activity. Significantly, the Cys-to-His mutant proteins retained nearly wild-type sequence-specific DNA-binding activity in the unmethylated state. These findings demonstrate that the three "spectator" Cys ligands communicate chemically with Cys 69 through the bound metal ion.
Sun
L J
LJ
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Yim
C K
CK
Verdine
G L
GL
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D015032:Zinc / Q000032:analysis
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; C436983:zinc tetrathiolate cluster; D004247:DNA; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D015032:Zinc; D003545:Cysteine