Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites.
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 degrees C it is 1.45microM 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 degrees C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5nM and 6.4nM, corresponding to Gibbs energies of -49kJmol(-1) and -46kJmol(-1), 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.
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Anatoly I
AI
Dragan
Leslie
L
Frank
Yingyun
Y
Liu
Elena N
EN
Makeyeva
Colyn
C
Crane-Robinson
Peter L
PL
Privalov
D051696:Activating Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001798:Blood Proteins / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D051790:G-Box Binding Factors; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D020673:Protein Renaturation; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014867:Water / Q000378:metabolism*
D051696:Activating Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001798:Blood Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D029701:Saccharomyces cerevisiae Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014867:Water; D004247:DNA; D011494:Protein Kinases