Abstract
The GAGA factor of Drosophila melanogaster uses a single Cys 2His 2-type zinc finger for specific DNA binding. The conformation and DNA binding mode of the GAGA zinc finger are similar to those of other structurally characterized zinc fingers. In almost all Cys 2His 2-type zinc fingers, the fourth position of the DNA-recognizing helix is occupied by the Leu residue involved in the formation of the minimal hydrophobic core. However, no systematic study on the precise role of the Leu residue in the hydrophobic core formation and DNA binding function has been reported. In this study, the Leu residue is substituted with other aliphatic amino acids having different side chain lengths and hydrophobicities, namely, Ile, Val, Aib, and Ala. The metal binding properties were studied by UV-vis spectroscopy. The peptide conformations were examined by CD and NMR spectroscopies. Furthermore, the DNA binding ability was examined with a gel mobility shift assay. Though the Ile, Val, and Aib mutants exhibited conformations similar to those of the wild type, the DNA binding affinity decreased as the side chain length of the amino acid decreased. Interestingly, the Val mutant can bind to the cognate DNA, while Aib cannot, in spite of the similarity in their secondary structures based on the CD measurements. Variable-temperature NMR experiments clearly indicated differences in the stability of the hydrophobic core between the Val and Aib mutants. This study demonstrates that the bulkiness of the conserved aliphatic residue is important in the formation of the well-packed minimal hydrophobic core and proper ternary structure and that the hydrophobic core stabilization is apparently related to the DNA binding function of the GAGA zinc finger.
