Structural basis and functional significance of oriented Fos -Jun heterodimer binding.

Many eukaryotic transcription regulatory proteins form heterodimers that bind palindromic recognition sites. Such heterodimers may bind these symmetrical recognition sites in either two opposite orientations that are related by 180° rotation about the dimer axis. Reversal of the orientation of heterodimer binding can alter interactions with transcription factors that bind to adjacent regulatory elements. Thus, control of heterodimer binding orientation provides a potential mechanism for transcriptional regulation. To study the orientation of heterodimer binding, we have used heterodimers formed by the basic region/leucine zipper (bZIP) domains of Fos and Jun transcription factors as a model system. The Fos and Jun oncoproteins form heterodimers that bind to the palindromic AP-1 regulatory element (TGACTCA) in both orientations in the X-ray crystal structure of the Fos-Jun-AP-1 complex. Fos-Jun heterodimers bind to different AP-1 sites in opposite orientations. I have defined the determinants for oriented Fos-Jun heterodimer binding and demonstrated functional differences between heterodimers that favor opposite orientations of binding. Gel-based fluorescence resonance energy transfer (gelFRET) analysis revealed that the orientation of Fos-Jun heterodimer binding was determined by base pairs flanking the AP-1 site. Exchange of charged amino acid residues outside the DNA contact regions of Fos and Jun reversed the binding orientation. The opposite DNA bending properties of Fos and Jun determine the orientation of heterodimer binding through long-range electrostatic interactions that minimize the electrostatic free energy of the Fos-Jun-AP-1 complex. Cooperative binding by Fos-Jun and Nuclear Factor of Activated T cells 1 (NFATI) requires a specific orientation of heterodimer binding. The interaction with NFAT1 can reverse the orientation of heterodimer binding. Multicolor FRET analysis of the dynamics of Fos-Jun-NFAT1 complexes revealed that Fos-Jun heterodimers could reorient while remaining bound to DNA. The preferred orientation of heterodimer binding affected the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Thus, the orientation of Fos-Jun heterodimer binding can influence transcriptional activity by altering cooperative interactions with neighboring transcription factors.Ph.D.BiochemistryBiological SciencesBiophysicsMolecular biologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123260/2/3068946.pd