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Structure-function analysis of the Z-DNA-binding domain Zalpha of dsRNA adenosine deaminase type I reveals similarity to the (alpha + beta) family of helix-turn-helix proteins.

ABSTRACT: RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins. An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zalpha and Zbeta, the function of which is as yet unknown. In this study, multidimensional NMR spectroscopy was used to show that the topology of Zalpha is alpha1beta1alpha2alpha3beta2beta3. Long-range NOEs indicate that beta1 and beta3 interact with each other. Site-directed mutagenesis was used to identify residues in alpha3, beta3 and the loop connecting beta2 to beta3 that affect Z-DNA binding. Also identified were 11 hydrophobic residues that are essential for protein stability. Comparison with known structures reveals some similarity between Zalpha and (alpha + beta) helix-turn-helix proteins, such as histone 5 and the family of hepatocyte nuclear factor-3 winged-helix-turn-helix transcription factors. Taken together, the structural and functional data suggest that recognition of Z-DNA by Zalpha involves residues in both the alpha3 helix and the C-terminal beta-sheet.

SUBMITTER: Schade M

PROVIDER: S-EPMC1171140 | biostudies-other | 1999 Jan

REPOSITORIES: biostudies-other

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Structure-function analysis of the Z-DNA-binding domain Zalpha of dsRNA adenosine deaminase type I reveals similarity to the (alpha + beta) family of helix-turn-helix proteins.

Schade M M Turner C J CJ Lowenhaupt K K Rich A A Herbert A A

The EMBO journal 19990101 2

RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins. An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zalpha and Zbeta, the function of which is as yet unknown. In this study, multidimensional NMR spectroscopy was used to show that the topology of Zalpha is alpha1beta1alpha2alpha3beta2beta3. Long-range NOEs indica ...[more]

PMID: 9889202

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Project description:Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

Dataset Information

Structure-function analysis of the Z-DNA-binding domain Zalpha of dsRNA adenosine deaminase type I reveals similarity to the (alpha + beta) family of helix-turn-helix proteins.

Publications

Structure-function analysis of the Z-DNA-binding domain Zalpha of dsRNA adenosine deaminase type I reveals similarity to the (alpha + beta) family of helix-turn-helix proteins.

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