Mechanisms of activation of interferon regulator factor 3: the role of C-terminal domain phosphorylation in IRF-3 dimerization and DNA binding.

The interferon regulatory transcription factor (IRF-3) is activated by phosphorylation of Ser/Thr residues clustered in its C-terminal domain. Phosphorylation of these residues, which increases the negative charge of IRF-3, results in its dimerization and association with DNA, despite the increase in repulsive electrostatic interactions. To investigate this surprising effect, the dimerization of IRF-3 and two phosphomimetic mutants, 2D (S396D, S398D) and 5D (S396D, S398D, S402D, T404D and S405D), and their binding to single-site PRDI and double-site PRDIII-PRDI DNA sequences from the IFN-beta enhancer have been studied.

Forces driving the binding of homeodomains to DNA.

Homeodomains are helix-turn-helix type DNA-binding domains that exhibit sequence-specific DNA binding by insertion of their "recognition" alpha helices into the major groove and a short N-terminal arm into the adjacent minor groove without inducing substantial distortion of the DNA. The stability and DNA binding of four representatives of this family, MATalpha2, engrailed, Antennapedia, and NK-2, and truncated forms of the last two lacking their N-terminal arms have been studied by a combination of optical and microcalorimetric methods at different temperatures and salt concentrations. It was found that the stability of the free homeodomains in solution is rather low and, surprisingly, is reduced by the presence of the N-terminal arm for the Antennapedia and NK-2 domains.

Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3.

The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.

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.

DNA-binding domain of GCN4 induces bending of both the ATF/CREB and AP-1 binding sites of DNA.

The interaction of proteins with DNA results, in some cases, in DNA bending, and this might have functional importance. However, when the protein-induced bending of DNA is small, its measurement presents a problem. It is shown that the fluorescence resonance energy transfer between fluorophores placed on the ends of the specially designed U-shaped DNA, which contains the DNA-binding sites at its central part, can be successfully used for this purpose.