Long-range electrostatic interactions influence the orientation of fos-jun binding at AP-1 sites

Abstract

Heterodimeric transcription regulatory proteins that bind palindromic DNA sequences can potentially bind their recognition sites in two opposite orientations. The orientation of transcription factor binding can control transcriptional activity by altering interactions with proteins that bind to adjacent regulatory elements. Fos-Jun heterodimers bind to AP-1 sites with different flanking sequences in opposite orientations. A gel-based fluorescence resonance energy transfer assay, gelFRET, was used to define the mechanism whereby amino acid residues and nucleotide base-pairs outside the Fos-Jun-AP-1 contact interface determine the orientation of heterodimer binding. Exchange of three amino acid residues adjacent to the basic DNA contact regions between Fos and Jun reversed the binding orientation. The effects of these amino acid residues on the orientation of heterodimer binding depended on base-pairs flanking the core AP-1 recognition sequence. Single amino acid and base-pair substitutions had parallel effects on DNA bending by Fos-Jun-AP-1 complexes and on heterodimer orientation. The binding orientation exhibited a close correspondence with both the difference in bending propensities of opposite sides of the AP-1 site as well as the difference in bending potentials of the Fos and Jun subunits of the heterodimer. The influence of flanking DNA sequences on heterodimer orientation was attenuated in the presence of high concentrations of multivalent cations. Base substitutions up to one helical turn from the center of the AP-1 site affected the binding orientation. Modification of flanking base-pairs with positively or negatively charged functional groups had opposite effects on the orientation of heterodimer binding. These changes in DNA charge had converse effects on the orientation preferences of heterodimers in which charged amino acid residues adjacent to the basic regions were exchanged between Fos and Jun. These results indicate that the orientation of heterodimer binding is determined primarily by minimization of the electrostatic free energy of the Fos-Jun-AP-1 complex. Consequently, long-range electrostatic interactions influence the architecture of nucleoprotein complexes.