Addition of Positively Charged Tripeptide to the N-terminus of Fos bZIP Domain, Implications on DNA Bending, Affinity and Specificity

grantor: University of TorontoGKH-Fos/Jun is a hybrid protein designed to contain a metal binding motif in the form of a GKH tripeptide at the N-terminus of Fos bZIP domain dimerized with the Jun bZIP domain. We examined the effect of the addition of positively charged GKH motif to Fos on the DNA binding characteristics of the Fos/Jun heterodimer. Comparison of the apparent dissociation constants of the unmodified control Fos/Jun (cFos/Jun) and GKH-Fos/Jun heterodimers reveals that GKH-Fos/Jun binds the AP-1 site with a 6 fold decreased affinity than does cFos/Jun. In addition, GKH-Fos/Jun exhibits a 4 fold increased affinity for nonspecific DNA in comparison to cFos/Jun. Furthermore, helical phasing analysis indicates that GKH-Fos/Jun and cFos/Jun both bend the DNA at the AP-1 site. However, the degree of the induced bend by GKH-Fos/Jun is greater than that induced by cFos/Jun. Our results suggest that the unfavorable energetic cost of increased DNA bending by GKH-Fos/Jun results in a decrease in specificity and affinity of binding of the heterodimer to the AP-1 site.M.Sc