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.     

The effect of C-terminal helix stabilization on specific DNA binding by monomeric GCN4 peptides.

DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25. Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25 C in an aqueous buffer containing 100 mm NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide-DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1:1 complex with a dissociation constant of 1.41 +/- 0.22 microm. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 +/- 0.09 microm for peptide br-C and 3.9 +/- 0.5 microM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the alpha-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations.     

Bacterial expression and characterization of the CREB bZip module: circular dichroism and 2D 1H-NMR studies.

In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259-327, purified to near homogeneity, maintains the sequence-specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259-327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259-327 for the CRE site was determined by gel retardation assays to be on the order of 10(-7) M. We employed CD spectroscopy to study the folding properties of the native and modified CREB259-327. The CD analyses of the native/modified CREB259-327 peptide demonstrated a 20% increase in the alpha-helical content upon binding to the cAMP response-element. Only a 5% increase in the alpha-helical content of CREB259-327 is observed upon binding to the AP-1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347, 575-578). In addition, the two-dimensional (2D) 1H-NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.     

A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.

The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.     

Calorimetric analysis of binding of two consecutive DNA strands to RecA protein illuminates mechanism for recognition of homology

RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (DeltaH) with temperature (heat capacity change, DeltaC(p)). In the presence of the ATP analogue ATPgammaS, the DeltaH for the binding of the first DNA strand depends upon temperature (large DeltaC(p)) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced DeltaC(p), indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the DeltaH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the DeltaH is larger than that for non-complementary DNA strand, but less than the DeltaH of the annealing of the complementary DNA without RecA. This small DeltaH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The DeltaH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.     

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.     

Energetics of sequence-specific protein-DNA association: binding of integrase Tn916 to its target DNA

The DNA binding domain of the transposon Tn916 integrase (INT-DBD) binds to its DNA target site by positioning the face of a three-stranded antiparallel beta-sheet within the major groove. Binding of INT-DBD to a 13 base pair duplex DNA target site was studied by isothermal titration calorimetry, differential scanning calorimetry, thermal melting followed by circular dichroism spectroscopy, and fluorescence spectroscopy. The observed heat capacity change accompanying the association reaction (DeltaC(p)) is temperature-dependent, decreasing from -1.4 kJ K(-1) mol(-1) at 4 degrees C to -2.9 kJ K(-1) mol(-1) at 30 degrees C. The reason is that the partial molar heat capacities of the free protein, the free DNA duplex, and the protein-DNA complex are not changing in parallel when the temperature increases and that thermal motions of the protein and the DNA are restricted in the complex. After correction for this effect, DeltaC(p) is -1.8 kJ K(-1) mol(-1) and temperature-independent. However, this value is still higher than DeltaC(p) of -1.2 kJ K(-1) mol(-1) estimated by semiempirical methods from dehydration of surface area buried at the complex interface. We propose that the discrepancy between the measured and the structure-based prediction of binding energetics is caused by incomplete dehydration of polar groups in the complex. In support, we identify cavities at the interface that are large enough to accommodate approximately 10 water molecules. Our results highlight the difficulties of structure-based prediction of DeltaC(p) (and other thermodynamic parameters) and emphasize how important it is to consider changes of thermal motions and soft vibrational modi in protein-DNA association reactions. This requires not only a detailed investigation of the energetics of the complex but also of the folding thermodynamics of the protein and the DNA alone, which are described in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3492-3502].     

Effect of monomeric and oligomeric sugar osmolytes on ΔGD, the Gibbs energy of stabilization of the protein at different pH values: Is the sum effect of monosaccharide individually additive in a mixture?

Thermal denaturation curves of ribonuclease-A were measured by monitoring changes in the far-UV circular dichroism (CD) spectra in the presence of different concentrations of six sugars (glucose, fructose, galactose, sucrose, raffinose and stachyose) and mixture of monosaccharide constituents of each oligosaccharide at various pH values in the range of 6.0-2.0. These measurements gave values of T(m) (midpoint of denaturation), DeltaH(m) (enthalpy change at T(m)), DeltaC(p) (constant-pressure heat capacity change) under a given solvent condition. Using these values of DeltaH(m), T(m) and DeltaC(p) in appropriate thermodynamic relations, thermodynamic parameters at 25 degrees C, namely, DeltaG(D)(o) (Gibbs energy change), DeltaH(D)(o) (enthalpy change), and DeltaS(D)(o) (entropy change) were determined at a given pH and concentration of each sugar (including its mixture of monosaccharide constituents). Our main conclusions are: (i) each sugar stabilizes the protein in terms of T(m) and DeltaG(D)(o), and this stabilization is under enthalpic control, (ii) the protein stabilization by the oligosaccharide is significantly less than that by the equimolar concentration of the constituent monosaccharides, and (iii) the stabilization by monosaccharides in a mixture is fully additive. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of protein in their native and denatured states are not perturbed on the addition of sugars.