DESIGN OF SEQUENSE-SELECTIVE DNA-BINDING SMALL PROTEINS

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We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4, and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain, and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. Addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon the DNA binding. CD spectral and thermodynamic studies indicate that the DNA -contacting region is folded in the presence or absence of DNA, and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of the basic leucine zipper proteins.Another design strategy utilized a well-folded small domain of C2H2 zinc finger motif as scaffold for the DNA binding α-helix. Appropriate substitution of the α-helical portion of the C2H2 zinc finger motif with amino acid residues necessary for the sequence-selective binding of GCN4 would afford a novel DNA binding domain with a folded structure. Studies on struacural aspects and the sequence-specific DNA binding of the novel protein in the presence of various metal ions are currently underway.