Jun DNA-binding is modulated by mutations between the leucines or by direct interaction of fos with the TGACTCA sequence

Mutations between the leucines of the "leucine zipper" domain of Jun D can either decrease (Asn 301 to Ala) or increase (Thr 307, Ala 308, to Glu, Val) homodimer formation and specific binding to DNA even though such changes do not modify the predicted alpha-helical structure of this region. As shown previously, addition of Fos strongly increases the affinity of Jun for DNA by forming a heterodimer. The jun down mutation (Asn 301 to Ala) also diminishes DNA binding by the Fos-Jun D heterodimer. These data strongly support the coiled coil conformation of this region where residues adjacent to the leucines are also important for dimer formation. Ultraviolet cross-linking experiments have shown that both Fos and Jun directly contact the TGACTCA palindromic sequence defined as a TPA (12-O-tetradecanoyl phorbol-13-acetate) response element or TRE. Both Jun homodimers and Jun-Fos heterodimers bind this TRE as well as the cAMP responsive element (CRE or TGACGTCA) with comparable affinities. While strong c-Jun or Jun D binding requires a perfect palindrome, Jun-Fos complexes can also efficiently recognize sequences where the right half of the palindrome is less conserved (TGACTAA or TGACGCA).     

Dimerization of leucine zippers analyzed by random selection.

The leucine zipper is a coiled coil that mediates specific dimerization of bZIP DNA-binding domains. A hydrophobic spine involving the conserved leucines runs down the coiled-coil and is thought to stabilize the dimer. We used the method of random selection to further define the primary sequence requirements for homodimer formation and heterodimer formation with Fos. When positions on either side of the hydrophobic spine of GCN4 are diversified to include the corresponding residues of Jun, a large percentage of the resulting sequences form homodimers, and a large percentage form heterodimers with Fos. Basic residues were preferred, but not essential, at position e of zippers which heterodimerize with Fos. When random sequences containing 5 heptad repeat of leucines are subject to a selection for homodimer formation, a diverse set of sequences is isolated. Certain residues are preferred at each position in the heptad repeat, although no essential primary sequence determinants could be identified. No pair of residues not involving the conserved leucines could be identified which strongly promotes homodimerization. These results suggest that factors determining leucine zipper dimerization are complex, with numerous interactions contributing to the association.     

Stabilities of leucine zipper dimers estimated by an empirical free energy method.

The leucine zipper motif is a characteristic amino acid sequence found in dimeric DNA-binding proteins. Computer-generated models for leucine zippers were constructed as alpha-helical coiled dimers with leucine repeated every seventh residue. An empirical Gibbs free energy, delta G, function which incorporates hydrophobic force, electrostatic interactions, and conformational entropy loss as the major intermolecular interactions was used to estimate the delta G of dimer formation in fos, jun, and GCN4 zipper sequences. The calculations showed that complexes known to form stable homo- or heterodimers have favorable (negative) delta G, while other less stable complexes have unfavorable (positive) delta G. Leucines in position d of the coiled coil contribute large hydrophobic stabilization energies while residues in the a position contribute less to dimer stability. Hydrophobic contributions show little sequence specificity, however, and do not contribute significantly to homo/heterodimer preference. Charged residues in the e and g positions, on the other hand, determine homo/heterodimer specificity. In GCN4 homodimers, residues GLU el, Glu b2, Lys g2, and Lys e4 greatly contribute to dimer stability. The preferential stability of fos-jun heterodimer over the jun-jun and fos-fos homodimers is primarily due to the side chains Asp b1, Glu g1, Asp b2, Glu e2, Glu g2, Glu g3, and Lys a5 of the fos helix, and Arg c1, Lys g1, Lys b2, Lys e2, Arg e4, and Glu g4 of the jun helix.     

The basic region of Fos mediates specific DNA binding.

The DNA-binding domains of the members of the Fos and Jun families of proteins consist of a basic region followed by a dimerizing segment with heptad repeats of leucine. Fos-Jun heterodimers and Jun alone, but not Fos alone, bind to the symmetrical sequences TGACTCA (AP-1 site) or TGACGTCA (cAMP response element or CRE). We set out to test the hypothesis that in the Fos-Jun heterodimer the basic region of Fos confers specific DNA-binding properties equivalent to the contribution of the basic region of Jun. Fos-Jun chimeric proteins were prepared consisting of the basic region of one protein joined to the leucine repeat of the other. Heterodimers with mixed Fos and Jun leucine repeat segments showed high affinity binding to the AP-1 site or CRE whether they contained two basic regions from Jun, two basic regions from Fos, or one from each source. Heterodimers with two Fos basic regions showed somewhat greater affinity for the CRE and AP-1 site than the heterodimer with two Jun basic regions. The DNA sequence specificity and the purine and phosphate DNA contact sites for each heterodimer were similar. We conclude that in the Fos-Jun heterodimer the basic region of Fos contributes specific DNA-binding properties equivalent to those of Jun. Our results support a model in which the Fos and Jun basic regions of the Fos-Jun heterodimer each interact with symmetrical DNA half sites.     

Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity

Association of Fos and Jun with the AP-1 site results in a conformational change in the basic amino acid regions that constitute the DNA-binding domain. We show that Fos and Jun induce a corresponding alteration in the conformation of the DNA helix. Circular permutation analysis indicated that both Fos-Jun heterodimers and Jun homodimers induce flexure at the AP-1 site. Phasing analysis demonstrated that Fos-Jun heterodimers and Jun homodimers induce DNA bends that are directed in opposite orientations. Fos-Jun heterodimers bend DNA toward the major groove, whereas Jun homodimers bend DNA toward the minor groove. Fos and Jun peptides encompassing the dimerization and DNA-binding domains bend DNA in the same orientations as the full-length proteins. However, additional regions of both proteins influence the magnitude of the DNA bend angle. Thus, despite the amino acid sequence similarity in the basic region Fos-Jun heterodimers and Jun homodimers form topologically distinct DNA-protein complexes.     

Comparison of in vivo selection and rational design of heterodimeric coiled coils

To investigate how electrostatic interactions restrict the associations of coiled coils, we improved a heterodimeric coiled coil (WinZip-A1B1) by in vivo selection and, alternatively, by rational design. Selection from libraries encoding variable edge (g and e) residues enriched g/e' ion pairs, but the optimum selected heterodimers unexpectedly retained two predicted repulsive g/e' pairs. The best genetically selected heterodimer displayed similar thermodynamic stability and specificity as a rationally designed dimer with predicted ion pairs at all edge positions. This rationally designed pair, however, was less effective than the best genetically selected pair in mediating dimerization in vivo. Thus, the effects of predicted charge pairs depend on sequence context, and complementary charges at the edge positions rationalize only a fraction of the sequences that form stable, specific coiled coils.