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The solution structure of the methylated form of the N-terminal 16-kDa domain of Escherichia coli Ada protein.

ABSTRACT: The N-terminal 16-kDa domain of Escherichia coli Ada protein (N-Ada16k) repairs DNA methyl phosphotriester lesions by an irreversible methyl transfer to its cysteine residue. Upon the methylation, the sequence-specific DNA binding affinity for the promoter region of the alkylation resistance genes is enhanced by 10(3)-fold. Then, it acts as a transcriptional regulator for the methylation damage. In this paper, we identified the methyl acceptor residue of N-Ada16k and determined the solution structure of the methylated form of N-Ada16k by using NMR and mass spectrometry. The results of a 13C-filtered 1H-13C HMBC experiment and MALDI-TOF MS and MS/MS experiments clearly showed that the methyl acceptor residue is Cys38. The solution structure revealed that it has two distinct subdomains connected by a flexible linker loop: the methyltransferase (MTase) subdomain with the zinc-thiolate center, and the helical subdomain with a helix-turn-helix motif. Interestingly, there is no potential hydrogen bond donor around Cys38, whereas the other three cysteine residues coordinated to a zinc ion have potential donors. Hence, Cys38 could retain its inherent nucleophilicity and react with a methyl phosphotriester. Furthermore, the structure comparison shows that there is no indication of a remarkable conformational change occurring upon the methylation. This implies that the electrostatic repulsion between the negatively charged DNA and the zinc-thiolate center may avoid the contact between the MTase subdomain and the DNA in the nonmethylated form. Thus, after the Cys38 methylation, the MTase subdomain can bind the cognate DNA because the negative charge of the zinc-thiolate center is reduced.

SUBMITTER: Takinowaki H

PROVIDER: S-EPMC2249770 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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The solution structure of the methylated form of the N-terminal 16-kDa domain of Escherichia coli Ada protein.

Takinowaki Hiroto H Matsuda Yasuhiro Y Yoshida Takuya T Kobayashi Yuji Y Ohkubo Tadayasu T

Protein science : a publication of the Protein Society 20060201 3

The N-terminal 16-kDa domain of Escherichia coli Ada protein (N-Ada16k) repairs DNA methyl phosphotriester lesions by an irreversible methyl transfer to its cysteine residue. Upon the methylation, the sequence-specific DNA binding affinity for the promoter region of the alkylation resistance genes is enhanced by 10(3)-fold. Then, it acts as a transcriptional regulator for the methylation damage. In this paper, we identified the methyl acceptor residue of N-Ada16k and determined the solution stru ...[more]

PMID: 16452614

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Project description:Alkylation damage to DNA occurs when cells encounter alkylating agents in the environment or when active alkylators are generated by nitrosation of amino acids in metabolic pathways. To cope with DNA alkylation damage, cells have evolved genes that encode proteins with alkylation-specific DNA repair activities. It is notable that these repair systems are conserved from bacteria to humans. In Escherichia coli, cells exposed to a low concentration of an alkylating agent, such as N-methyl-Nâ-nitro-N-nitrosoguanidine (MNNG) or methyl methanesulfonate (MMS), show a remarkable increase in resistance to both the lethal and mutagenic effects of subsequent high-level challenge treatments with the same or other alkylating agents. This increased resistance has been known as âadaptive responseâ to alkylation damage in DNA. To date, four genes have been identified as components of this response, ada, alkA, alkB and aidB. The ada gene encodes the Ada protein, which has the dual function of a transcriptional regulator for the genes involved in the adaptive response, and a methyltransferase that demethylates two methylated bases (O6meG and O4meT) and methylphosphotriesters produced by methylating agents in the sugar phosphate backbone. The differences between the wild-type and mutant strains were characterized at transcriptome levels. In addition, the global changes in gene expressions in response to alkylating agents (MMS), in E. coli K-12 W3110 and ada mutant strains were also analyzed. The analysis of time- and strain-dependent adaptive responses revealed the regulatory and physiological characteristics of the Ada-dependent adaptive response in E. coli. In order to examine the intracellular changes that are induced by the ada gene deletion in the MMS-untreated, normal growth condition, the expression levels of genes of ada mutant cells were compared with those of wild-type cells at the mid-log growth phase (at 0.5 h sampling point). Cells were cultivated at 37oC and 250 rpm in 100 mL of Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) in 250-mL Erlenmeyer flasks. Transcriptome analysis were also performed for the samples (E. coli wildtype and ada mutant strains) taken at 0.5, 1.5 and 3.9 h following MMS treatment for both MMS-treated and -untreated control cultures, and the expression levels were compared.

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The solution structure of the methylated form of the N-terminal 16-kDa domain of Escherichia coli Ada protein.

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The solution structure of the methylated form of the N-terminal 16-kDa domain of Escherichia coli Ada protein.

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