Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF domain) in plant

Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pM range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.