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

被引:24
作者
Lin, YX
Dötsch, V
Wintner, T
Peariso, K
Myers, LC
Penner-Hahn, JE
Verdine, GL
Wagner, G
机构
[1] Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA
[2] Harvard Univ, Dept Chem & Biol Chem, Cambridge, MA 02138 USA
[3] Harvard Univ, Dept Phys, Cambridge, MA 02138 USA
[4] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
关键词
D O I
10.1021/bi002109p
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Escherichia coli protein Ada specifically repairs the S-p diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center. The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes. Ada thus acts as a chemosensor to activate repair 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 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer. To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated. Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada. The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA, This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group.
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收藏
页码:4261 / 4271
页数:11
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