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. It was found that: ( a) the mutations in the C- terminal domain do not affect the state of the DNA-binding N- terminal domain or its ability to bind target DNA; ( b) in the 5D-mutant, the local increase of negative charge in the C- terminal domain induces restructuring, resulting in the formation of a stable dimer; ( c) dimerization of IRF-3 is the basis of its strong binding to PRDIII - PRDI sites since binding of 5D to the single PRDI site is similar to that of inactivated IRF-3. Analysis of the binding characteristics leads to the conclusion that binding of dimeric IRF-3 to the DNA with two tandem-binding sites, which are twisted by similar to 1008 relative to each other, requires considerable work to untwist and/ or bend the DNA.