Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: a comparison to Homo sapiens B-ZIP motifs

Abstract

Basic region-leucine zipper (B-ZIP) proteins are a class of dimeric sequence-specific DNA-binding proteins unique to eukaryotes. We have identified 67 B-ZIP proteins in the Arabidopsis thaliana genome. No A.thaliana B-ZIP domains are homologous with any Homo sapiens B-ZIP domains. Here, we predict the dimerization specificity properties of the 67 B-ZIP proteins in the A.thaliana genome based on three structural properties of the dimeric alpha-helical leucine zipper coiled coil structure: (i) length of the leucine zipper, (ii) placement of asparagine or a charged amino acid in the hydrophobic interface and (iii) presence of interhelical electrostatic interactions. Many A.thaliana B-ZIP leucine zippers are predicted to be eight or more heptads in length, in contrast to the four or five heptads typically found in H.sapiens, a prediction experimentally verified by circular dichroism analysis. Asparagine in the a position of the coiled coil is typically observed in the second heptad in H.sapiens. In A.thaliana, asparagine is abundant in the a position of both the second and fifth heptads. The particular placement of asparagine in the a position helps define 14 families of homodimerizing B-ZIP proteins in A.thaliana, in contrast to the six families found in H.sapiens. The repulsive interhelical electrostatic interactions that are used to specify heterodimerizing B-ZIP proteins in H.sapiens are not present in A.thaliana. Instead, we predict that plant leucine zippers rely on charged amino acids in the a position to drive heterodimerization. It appears that A.thaliana define many families of homodimerizing B-ZIP proteins by having long leucine zippers with asparagine judiciously placed in the a position of different heptads.

Figure 1

X-ray structure of GCN4 B-ZIP motif bound to double stranded DNA (1). The N-terminal of the protein, the basic region and leucine zipper are labeled. The first three heptads of the leucine zipper are delineated.

Figure 2

Amino acid sequence of the 67 A.thaliana B-ZIP domains. Proteins are arranged into families, A–T shown in bold in the first column, with similar predicted dimerization properties. The second column depicts names of the families from Jakoby et al. (7). The solid line delineates homodimerizing proteins (A–N) from proteins that have complex dimerization pattern (P–T). The leucine zipper region is divided into heptads (gabcdef) to help visualize the g↔e′ pairs. Amino acids predicted to regulate dimerization specificity are color-coded. If the g and following e positions contain charged amino acids we colored the heptad from g to the following e. We use four colors to represent g↔e′ pairs. Green is for the attractive basic-acidic pairs (R↔E and K↔E), orange is for the attractive acidic-basic pairs (E↔R, E↔K, D↔R and D↔K), red is for repulsive acidic pairs (E↔E and E↔D), and blue is for repulsive basic pairs (K↔K and R↔K). If only one of the two amino acids in the g↔e′ pair is charged, we color that residue blue for basic and red for acidic. If the a or d position is polar, it is colored black and if either is charged, it is colored purple. The prolines and glycines are colored red to indicate a potential break in the α-helical structure. The predicted C-terminal boundary of the B-ZIP leucine zipper is denoted by an asterisk that enables us to define the frequencies of amino acids in different position of the leucine zipper. The C-terminal boundary is defined by the presence hydrophobic amino acids in the a and d positions, charged amino acids in the e and g positions, the absence of proline or pairs of glycines anywhere in the structure, and the absence of charged amino acids in the a and d positions. In the majority of cases, the decision was straight forward. However, in several instances, it is more ambiguous. For example, we indicate that the DPBF3 and DPBF4 leucine zipper stops after the second heptad because the third heptad has a K and E in the a and d positions, respectively; which should prevent leucine zipper formation. For the same reason At5g44080 and GBF4 stops after the third heptad as they have a R and K in the a and d positions, respectively, in the fourth heptad. At1g06070 goes to the ninth heptad even though it has two glycines in the fourth heptad. We would normally think this would terminate a leucine zipper structure but it appears very canonical from the fifth to the ninth heptads so be propose that it continues through the fourth heptad. This type of thinking was used to define all C-terminal boundary of all the leucine zippers. We do not intend these definitions of the C-terminus to be definitive, only approximate. The natural C-terminus is denoted with circumflex accent. The protein sequence for the tenth heptad of posF21, an N family member, and At2g13150, a P family member, that are predicted to not form a coiled-coil is LTGQVAP and VLISNEK, respectively. The dot in front of a protein indicates that it has been experimentally shown to form a homodimer.

Figure 3

Pie chart presenting the frequency of amino acids in all the g, e, a and d positions of the leucine zipper for both H.sapiens and A.thaliana B-ZIP proteins.

Figure 4

Histogram of the frequency of asparagine in the a positions of the leucine zippers for all H.sapiens and A.thaliana B-ZIP proteins.

Figure 5

Histogram of the frequency of attractive or repulsive g↔e′ pairs per heptad for both H.sapiens and A.thaliana B-ZIP proteins.

Figure 6

(A) Circular dichroism spectra from 200–260 nm of CREB and Opaque, 4 μM at 6°C. The asterisks indicate the minima at 222 nm at which the thermal denaturations of these two proteins were monitored. (B) Thermal denaturation curves of CREB and Opaque at 4 μM concentration monitored at 222 nm.