Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites

A. Dragan, L. Frank, Y. Liu, E. Makeyeva, Colyn Crane-Robinson, P. Privalov

    Research output: Contribution to journalArticlepeer-review

    Abstract

    The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry. The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 8C it is 1.45 mM and increases with temperature. To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP. The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments. Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 8C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5 nM and 6.4 nM, corresponding to Gibbs energies of K49 kJ molK1 and K46 kJ molK1, respectively. Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites. In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AP-1 and ATF/CREB sites, and correspondingly the entropies of association, are very different. bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AP-1 complex incorporates significantly more water molecules than the ATF/CREB complex.
    Original languageEnglish
    Pages (from-to)865-878
    Number of pages14
    JournalJournal of Molecular Biology
    Volume343
    Issue number4
    DOIs
    Publication statusPublished - 29 Oct 2004

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