DNA-Binding ability of GAGA zinc finger depends on the nature of amino acids present in the beta-hairpin.
The GAGA factor of Drosophila melanogaster uses a single Cys2-His2-type zinc finger for specific DNA binding. Comparative sequence alignment of the GAGA zinc finger core with other structurally characterized zinc fingers reveals that the beta-hairpin of the GAGA zinc finger prefers amino acids with an aliphatic side-chain different from those of other zinc fingers. To probe the substitution effect of aromatic amino acids in the beta-hairpin on the DNA binding, three mutant peptides were designed by substituting consensus phenylalanine, an aromatic amino acid, at key positions in the beta-hairpin region. The metal-binding and the overall fold of the mutant peptides are very similar to those of the wild-type as shown by UV-vis absorption spectroscopy and circular dichroism spectroscopy. However, the gel mobility shift assay and isothermal calorimetric studies demonstrated that none of the mutants are able to bind the cognate DNA substrate, although the mutation is confined only to the beta-hairpin region. The present results suggest that the nature of the amino acids in the beta-hairpin plays an important role in the DNA-binding of the GAGA factor protein.
Dhanasekaran
Muthu
M
Faculty of Pharmaceutical Sciences, Doshisha Women's University, Koudo, Kyotanabe-Shi 610-0395, Japan.
Negi
Shigeru
S
Imanishi
Miki
M
Sugiura
Yukio
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D004331:Drosophila melanogaster / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D010649:Phenylalanine