Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus.

Abstract

GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.