B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners

Abstract

The basic region-leucine zipper (B-ZIP) (bZIP) protein motif dimerizes to bind specific DNA sequences. We have identified 27 B-ZIP proteins in the recently sequenced Drosophila melanogaster genome. The dimerization specificity of these 27 B-ZIP proteins was evaluated using two structural criteria: (1) the presence of attractive or repulsive interhelical g<-->e' electrostatic interactions and (2) the presence of polar or charged amino acids in the 'a' and 'd' positions of the hydrophobic interface. None of the B-ZIP proteins contain only aliphatic amino acids in the'a' and 'd' position. Only six of the Drosophila B-ZIP proteins contain a "canonical" hydrophobic interface like the yeast GCN4, and the mammalian JUN, ATF2, CREB, C/EBP, and PAR leucine zippers, characterized by asparagine in the second 'a' position. Twelve leucine zippers contain polar amino acids in the first, third, and fourth 'a' positions. Circular dichroism spectroscopy, used to monitor thermal denaturations of a heterodimerizing leucine zipper system containing either valine (V) or asparagine (N) in the 'a' position, indicates that the V-N interaction is 2.3 kcal/mole less stable than an N-N interaction and 5.3 kcal/mole less stable than a V-V interaction. Thus, we propose that the presence of polar amino acids in novel positions of the 'a' position of Drosophila B-ZIP proteins has led to leucine zippers that homodimerize rather than heterodimerize.

Figure 1

X-ray structure of GCN4 B-ZIP motif bound to a TRE DNA sequence (Ellenberger et al. 1992). The DNA is in red. The B-ZIP α-helices are in blue with the leucines in the ‘d’ position shown in gray. The N-terminus of the protein is labeled. The basic region and leucine zipper are labeled. The first three heptads of the leucine zipper are highlighted.

Figure 2

Rectangular cladogram representing the phylogenetic relationship among the Drosophila B-ZIP proteins and their closest human counterparts. The tree was made from a multiple alignment of 25 amino acids from the basic region and the first four heptads of the leucine zipper region.

Figure 3

Alignment of 27 identified Drosophila melanogaster B-ZIP motifs using the single letter amino acid code. The proteins are arranged into groups based on the number of attractive g↔e‘ interactions minus repulsive g↔e‘ interactions ranging from 3 to –2 pairs. The column starts with the name of the protein. Next is the name of the closest mammalian homolog, followed by the gi# for the Drosophila melanogaster sequence. The protein sequence of the B-ZIP motif follows. The number of amino acids from the predicted N terminus of the protein to the B-ZIP motif is given in parentheses. The C terminus of each sequence is either the natural C terminus denoted by an asterisk (*), or a truncation with the number of amino acids to the C terminus in parentheses. To help visualize the potential g↔e‘ interactions, we grouped heptads (gabcdef). If both the ‘g’ and ‘e’ positions contain charged amino acids, we color both of these amino acids and the intervening ones (gabcde). We use green for the attractive basic–acidic pairs (R↔E and K↔E, K↔D), orange for the attractive acidic–basic pairs (E↔R, E↔K, D↔K, and D↔R), red for the repulsive acidic pairs (E↔E, D↔E, and E↔D), and blue for the repulsive basic pairs (K↔K, R↔K, K↔R, and R↔R). If only one of the two amino acids in the g↔e‘ pair is charged, we color only that amino acid: red if it is acidic and blue if it is basic. If the ‘a’ or ‘d’ positions contain polar or charged amino acids, they are colored black. The α-helix breaking prolines, indicative of the C terminus of the leucine zipper, are colored red.

Figure 4

End view, looking from N terminus to C terminus, of a coiled coil with the seven unique positions of the heptad presented as ellipses. The ‘a’ and ‘d’ positions are colored black. The four possible combinations of acidic and basic amino acids in the ‘g’ and ‘e’ positions are presented and color coded as used in Figure 2. (A) An α-helix with a g↔e‘ pair containing an acidic amino acid in the ‘g’ position and a basic amino acid in the following ‘e’ position (orange in Fig. 2) can form a homodimer or heterodimer with a similarly charged α-helix. (B) An α-helix with a g↔e‘ pair containing a basic amino acid in the ‘g’ position and an acidic amino acid in the following ‘e’ position (green in Fig. 2) can form a homodimer or heterodimer with a similarly charged α-helix. (C) A heterodimer between an acidic g↔e‘ pair (red in Fig. 2) and a basic g↔e‘ pair (blue in Fig. 2). (D) A dimer with an “incomplete” g↔e‘ pair resulting in promiscuous dimerization.