The Linker of the Interferon Response Factor 3 Transcription Factor Is Not Unfolded

Abstract

Interferon response factor 3 (IRF-3) is a transcription factor that plays an essential role in controlling the synthesis of interferon-β (IFN-β) and is a protein consisting of two well-defined domains, the N-terminal DNA-binding and the C-terminal dimerization domains, connected by a 75-residue linker, supposedly unfolded. However, it was not clear whether in intact IRF-3 this linker segment of the chain, which carries the nuclear export signal and includes a region of high helical propensity, remains unfolded. This has been investigated using nuclear magnetic resonance by ligating the (15)N-labeled linker to the unlabeled N-terminal and C-terminal domains. It was found that, while the linker alone is indeed in a completely unfolded state, when ligated to the C-terminal domain it shows some ordering, and this ordering becomes much more pronounced when the linker is also ligated to the N-terminal domain. Thus, in intact IRF-3, the linker represents a folded structural domain; i.e., IRF-3 is a three-domain globular protein. Light scattering studies of wild-type IRF-3 showed that these three domains are tightly packed, and therefore, the dimer of IRF-3, which is formed upon phosphorylation of its C-terminal domains following virus invasion, must be a rather rigid and compact construction. One would then expect that binding of such a dimer to its tandem recognition sites PRDIII and PRDI, which are located on opposing faces of the IFN-β enhancer DNA, should result in deformation of the DNA. Analysis of the characteristics of binding of the monomeric and dimeric IRF-3 to the enhancer DNA indeed showed that formation of this complex requires considerable work for deformation of its components, most likely bending of the DNA. Such bending was confirmed by atomic force microscopy of dimeric IRF-3 bound to the PRDII-PRDI tandem recognition sites placed at the middle of a 300 bp DNA probe. Bending of DNA by IRF-3 must be significant in the assembly and function of the IFN-β enhancer.

Structural Studies of IRF4 Reveal a Flexible Autoinhibitory Region and a Compact Linker Domain

“…It shows that the linker region most likely adopts a folded conformation, where it may interact with the DBD and IAD domains located at either end of the molecule. This finding is supported by our CD studies and a report suggesting that the linker in IRF3 is not unfolded but may also adopt a folded conformation (58). Thus, our SAXS IRF4 envelope may represent the general domain architecture for all IRF proteins and suggest that the linker domain may play a role in the regulation of IRF function.…”

Glycogen synthase kinase 3 negatively regulates IFN regulatory factor 3 transactivation through phosphorylation at its linker region

“…Recent structural analysis indicated that the linker represents a folded structural domain. 28,29 Strong binding of the dimeric IRF3 to the PRDII–PRDI tandem recognition sites needs to bend DNA by about 100 o (Figure 5). Our current study shows that the phosphorylation status at Ser123, Ser173 and Thr180 residues of IRF3(5D) affects its transactivation activity.…”

“…The domain structure and activation mechanism of IRF3 are very similar to those of Smads. The structural analysis indicated dimeric IRF3 bound to the PRDII–PRDI tandem recognition sites cause a bending of DNA, 28,29 so it is possible that the phosphorylation of IRF3 linker region may reduce the flexibility required for the strong binding to the target DNA (Figure 5). Consequently, it may then affect the proper assembly of the transactivation complexes.…”

Pestivirus N pro Directly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

“…However, the full-length IRF3 structure is unavailable, and it is therefore not known how the DBD and the IAD are arranged in the IRF3 monomer and dimer (11,44,45). The linker region is shown to be partially helical in the full-length IRF3 protein, while it is not structured when either of the domains is absent (46). Thus, the intact linker region may also be involved in N pro binding.…”