Structural basis for the functional switch of the E-coli Ada protein

The Escherichia coli protein Ada specifically repairs the S-p diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer o f the methyl group to its own Cys 69 which is part of a zinc-thiolate cente r. The methyl transfer converts Ada into a transcriptional activator that b inds sequence-specifically to promoter regions of its own gene and other me thylation resistance genes. Ada thus acts as a chemosensor to activate repa ir mechanisms in situations of methylation damage. Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 d uring the repair process and provides a basis for understanding the mechani sm of the conformational switch triggered by methyl transfer. To further el ucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X -ray absorption near-edge structure) data were acquired, which confirmed th at the zinc-thiolate center is maintained when N-Ada is methylated. Thus, l igand exchange is not the mechanism that enhances sequence-specific DNA bin ding and transcriptional activation upon methylation of N-Ada. The mechanis m of the switch was further elucidated by recording NOESY spectra of specif ically labeled methylated-Ada/DNA complexes, which showed that the transfer red methyl group makes many contacts within N-Ada but none with the DNA, Th is implies that methylation of N-Ada induces a structural change, which enh ances the promoter affinity of a remodeled surface region that does not inc lude the transferred methyl group.