DNA Binding of Jun and Fos bZip Domains: Homodimers and Heterodimers Induce a DNA Conformational Change in Solution

Abstract

We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.

Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling

“…The K d of Fos homodimers in HeLa cells was 6.7 Ϯ 1.7 M, which is on the same order of magnitude as the value of 5.6 M determined for its isolated leucine zippers in vitro by circular dichroism (14). Values reported for the heterodimers of the isolated leucine zippers (10,48,49) or longer polypeptides (50) in vitro varied between 1 and 140 nM. For the Fos-Jun heterodimer, we found a K d range of 10 to 370 nM in live cells, which depended on the Fos/Jun ratio and on putting the donor and acceptor tags on one or the other protein.…”

“…In addition to forming stable heterodimers with c-Fos (7-9), c-Jun can also homodimerize, as revealed in vitro by electrophoretic mobility shift assay (EMSA) (8), and bind to DNA as a homodimer, although with lower affinity than the heterodimer (8,10). In contrast, the c-Fos homodimer was found to be unstable in vitro, and thus, c-Fos has been thought to interact with DNA only by forming heterodimers with c-Jun (9,11,12).…”

Protein–protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

“…Indeed, the c-Jun RhG /c-Jun Cy5 interactions both with and without non-labeled AP-1 oligonucleotide could be detected in this study, whereas the interaction among c-Jun RhG /Cy5-labeled AP-1/non-labeled Jun was not detected in the previous study (11). The apparent K d of c-Fos/c-Jun/AP-1 found in this study was in good agreement with reported values (25,26). The K d of c-Jun homodimer and AP-1 sequence also coincided with the value of 140 nM determined previously (25).…”

Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions

“…As shown in Figure 5C, only the Fos-Jun-DNA complex molecule carrying both RhG and Cy5 was observed in the cross-correlation and quantified, but not the Fos-Fos and Jun-Jun pairs. The apparent dissociation constant K d for the binding of the Fos-Jun heterodimer to DNA can be obtained from a single FCCS measurement (Földes-Papp and Kinjo 2001; Jankowski and Janka 2001): The K d value was found to be 30 nM (see supplemental data on the web site), which is consistent with a value of 50 nM independently derived from the results of gel shift assay (John et al 1996).…”