Table 1.
Experimentally determined thermodynamic parameters for the binding of bZIP domains of Jun-Fos heterodimer to dsDNA oligos containing the wild-type (WT) consensus motif TGACTCA and all possible single nucleotide variants thereof obtained from ITC measurements at 25°C and pH 8.0
| Motif | Sequence | n | Kd/μM | ΔH/kcalmol-1 | TΔS/kcalmol-1 | ΔG/kcalmol-1 | Gene Promoter |
|---|---|---|---|---|---|---|---|
| WT | TGACTCA | 1.02 ± 0.01 | 0.21 ± 0.01 | -35.08 ± 0.22 | -25.94 ± 0.19 | -9.12 ± 0.03 | Metallothionein 2A |
| A-3 | AGACTCA | 0.98 ± 0.09 | 3.55 ± 0.76 | -31.02 ± 0.59 | -23.56 ± 0.48 | -7.45 ± 0.13 | Aflatoxin B1 aldehyde reductase AKR7 |
| C-3 | CGACTCA | 0.97 ± 0.04 | 5.96 ± 2.28 | -19.70 ± 0.56 | -12.53 ± 0.32 | -7.16 ± 0.23 | Transcriptional activator ORF50 |
| G-3 | GGACTCA | 1.03 ± 0.06 | 2.82 ± 0.17 | -31.36 ± 0.36 | -23.80 ± 0.36 | -7.58 ± 0.04 | Mu-opioid receptor MOR1 |
| A-2 | TAACTCA | 0.98 ± 0.01 | 6.31 ± 0.08 | -26.48 ± 0.28 | -19.37 ± 0.30 | -7.10 ± 0.01 | Cytochrome P450 enzyme CYP27B1 |
| C-2 | TCACTCA | 1.05 ± 0.04 | 8.10 ± 1.06 | -30.74 ± 0.20 | -23.77 ± 0.27 | -6.95 ± 0.08 | Monocyte Chemotactic Protein MCP1 |
| T-2 | TTACTCA | 1.00 ± 0.02 | 2.08 ± 0.05 | -13.98 ± 0.14 | -6.23 ± 0.17 | -7.76 ± 0.01 | Transcriptional regulator JE/MCAF |
| C-1 | TGCCTCA | 0.92 ± 0.03 | 4.13 ± 0.59 | -29.89 ± 0.21 | -22.53 ± 0.30 | -7.35 ± 0.08 | Pro-opiomelanocortin hormone POMC |
| G-1 | TGGCTCA | 1.06 ± 0.01 | 3.90 ± 0.50 | -33.61 ± 0.13 | -26.21 ± 0.06 | -7.39 ± 0.08 | Bone protein osteocalcin |
| T-1 | TGTCTCA | 0.98 ± 0.01 | 3.62 ± 0.30 | -36.89 ± 0.06 | -29.46 ± 0.11 | -7.43 ± 0.05 | Transforming growth factor TGFB1 |
| A0 | TGAATCA | 0.95 ± 0.04 | 0.58 ± 0.04 | -39.88 ± 0.10 | -31.36 ± 0.06 | -8.52 ± 0.04 | Mitochondrial proton carrier UCP1 |
| G0 | TGAGTCA | 1.05 ± 0.04 | 0.14 ± 0.01 | -42.87 ± 0.16 | -33.52 ± 0.21 | -9.34 ± 0.03 | Cell cycle regulator cyclin D1 |
| T0 | TGATTCA | 1.04 ± 0.04 | 0.50 ± 0.02 | -38.85 ± 0.21 | -30.25 ± 0.21 | -8.60 ± 0.02 | Tyrosine hydroxylase TH |
| A+1 | TGACACA | 1.00 ± 0.03 | 0.92 ± 0.03 | -37.06 ± 0.14 | -28.82 ± 0.17 | -8.24 ± 0.02 | Glucocorticoid receptor GR |
| C+1 | TGACCCA | 0.95 ± 0.06 | 4.45 ± 0.40 | -31.95 ± 0.09 | -24.63 ± 0.02 | -7.31 ± 0.05 | Interferon IFN-γ |
| G+1 | TGACGCA | 0.95 ± 0.01 | 0.64 ± 0.01 | -32.78 ± 0.13 | -24.33 ± 0.15 | -8.46 ± 0.01 | ECM glycoprotein fibronectin 1 |
| A+2 | TGACTAA | 1.04 ± 0.04 | 2.00 ± 0.05 | -20.90 ± 0.09 | -13.11 ± 0.13 | -7.78 ± 0.01 | MAP kinase phosphatase MKP3 |
| G+2 | TGACTGA | 0.92 ± 0.03 | 5.92 ± 1.05 | -28.16 ± 0.19 | -21.01 ± 0.08 | -7.14 ± 0.11 | Steroidogenic acute regulator STAR |
| T+2 | TGACTTA | 1.07 ± 0.03 | 5.16 ± 0.15 | -27.88 ± 0.11 | -20.65 ± 0.08 | -7.22 ± 0.02 | Nerve growth Factor NGF |
| C+3 | TGACTCC | 1.04 ± 0.02 | 3.07 ± 0.29 | -31.90 ± 0.11 | -24.35 ± 0.04 | -7.53 ± 0.06 | Chloride channel CLC5 |
| G+3 | TGACTCG | 0.97 ± 0.02 | 1.80 ± 0.17 | -33.33 ± 0.06 | -25.46 ± 0.02 | -7.85 ± 0.06 | Amyloid precursor protein β-APP |
| T+3 | TGACTCT | 1.02 ± 0.03 | 3.30 ± 0.15 | -42.46 ± 0.13 | -34.97 ± 0.10 | -7.49 ± 0.03 | Tumor suppressor p53 |
Note that the DNA sequence shown for the TGACTCA motif and its single nucleotide variants corresponds to the sense strand only and nucleotides flanking these motifs have been omitted for clarity (see Figures 1b-d). The substituted nucleotide relative to the TGACTCA motif is underlined. One example of a gene promoter that contains a particular TGACTCA variant for recruiting the AP1 transcription factor is provided for physiological relevance (15-27). The values for the stoichiometry (n), affinity (Kd) and enthalpy change (ΔH) accompanying the binding of bZIP domains of Jun-Fos heterodimer to dsDNA oligos were obtained from the fit of a function, based on the binding of a ligand to a macromolecule using the law of mass action (41), to the ITC isotherms. Free energy of binding (ΔG) was calculated from the relationship ΔG=RTlnKd, where R is the universal molar gas constant (1.99 cal/mol/K) and T is the absolute temperature (K). Entropic contribution (TΔS) to binding was calculated from the relationship TΔS=ΔH-ΔG. Errors were calculated from 2-3 independent measurements. All errors are given to one standard deviation.