Coupled Folding and Site-Specific Binding of the GCN4-bZIP Transcription Factor to the AP-1 and ATF/CREB DNA Sites Studied by Microcalorimetry

Abstract

The site-specific interaction of the basic leucine zipper protein C62GCN4, which corresponds to the C-terminal sequence 220−281 of the yeast transcription factor GCN4, with the AP-1 and ATF/CREB DNA recognition sites was analyzed by isothermal titration microcalorimetry. Free C62GCN4 is a dimer composed of a C-terminal leucine zipper and a basic, mainly unstructured DNA binding domain. Upon association with the target DNA, C62GCN4 folds to a fully α-helical dimer [Ellenberger et al. (1992) Cell 71, 1223−1237; König and Richmond (1993) J. Mol. Biol. 233, 139−154]. The protein-bound AP-1 site is straight, and the protein-bound ATF/CREB site is bent by 20° toward the leucine zipper domain. The coupling between protein folding and DNA association resulting in two conformationally different complexes with C62GCN4 poses interesting thermodynamic problems. The association was strongly exothermic for both DNA target sites. The free energies of binding were indistinguishable in buffers of low salt concentration, and no change of the protonation state of C62GCN4 and/or the DNA target site occurred on formation of the complexes. Both complexes exhibited large and negative heat capacity changes. The empirical correlation between buried nonpolar and polar surfaces and the reduction in heat capacity concomitant to complexation did hold for the reaction with the AP-1 site at low salt concentration. However, in the case of the ATF/CREB site, the change in heat capacity was larger than could be accounted for by the burial of solvent-accessible surface. Potential sources of the extra decrement in the heat capacity could be restrictions in the vibrational modes of polar groups and of bound water molecules at the protein−DNA interface, thought to result from the bending of the ATF/CREB site. In the presence of high concentrations of glutamate and NaCl, the complex with the ATF/CREB site was significantly weaker than the complex with the AP-1 site.