title: Mechanisms of activation of interferon regulator factor 3: the role of C-terminal domain phosphorylation in IRF-3 dimerization and DNA binding.
abstract: 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 approximately 100 degrees relative to each other, requires considerable work to untwist and/or bend the DNA.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA. dragan@jhu.edu
authors.forename: Anatoly I
authors.initials: AI
authors.lastname: Dragan
authors.forename: Victoria V
authors.initials: VV
authors.lastname: Hargreaves
authors.forename: Elena N
authors.initials: EN
authors.lastname: Makeyeva
authors.forename: Peter L
authors.initials: PL
authors.lastname: Privalov
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D050838:Interferon Regulatory Factor-3 / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D050838:Interferon Regulatory Factor-3; D004247:DNA