The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
IRF-3 binding site
Functional site description:
The recognition of pathogen-associated molecular patterns (PAMPs) involves different pathways that can trigger convergent antimicrobial responses. In response to various microbial and viral infections, various innate adaptor proteins like STING, MAVS and TRIF trigger IRF-3 activation and production of type I interferons (IFNs) that are essential for host protection. IRF-3 protein is activated by binding to a conserved motif, referred to as pLxIS present in the adaptor proteins that are phosphorylated by TBK1 or IKKepsilon. Once phosphorylated, the motif binds to the transcription factor IRF-3 resulting in TBK1-dependent phosphorylation of an additional motif pLxIS in IRF-3. Phosphorylated IRF-3 forms a homodimer that activates the protein and positively regulates the transcription of IFN-beta. The rotavirus NSP1 protein also contains the pLxIS motif which binds to the same binding region in IRF-3 and is able to escape innate immune recognition by interfering with the IRF-3-dependent pathway.
ELM Description:
The innate immune response in mainly initiated by pathogen-responsive activation of the transcription factor IRF-3.IRF-3 activation is initiated when it binds to innate adaptor proteins like STING, MAVS and TRIF through a conserved phosphorylated motif, pLxIS where p represents a hydrophilic residue and S represents a phosphorylated serine. Moreover, IRF-3 itself contains the pLxIS motif where it is important for IRF-3 dimerization and activation. An alternative of the motif is found in rotavirus NSP1 protein which does not require the phosphorylated serine to mediate binding. In the eukaryotic examples, the mutation of this serine residue abolishes the interaction.
The motifs interact with the same ligand-binding surface of IRF-3, but the residues upstream of the motif are also important for effective binding even though they vary in the different proteins. A five-site binding model is seen in pNSP1-IRF-3 binding and in IRF-3 dimerization (Zhao,2016). Here, the positively charged patch around the IRF-3 Arg211 interacts with negatively charged residues or the phosphoserine upstream of the pLxIS motif, followed by binding sites for three hydrophobic residues within the motif. At the C-terminus, the positively charged cluster around Arg285 recognizes the phosphoserine of the pLxIS motif. Subsets of these five binding sites are involved in pSTING, pMAVS, pTRIF, and NSP1 binding and might account for their different affinity towards IRF-3.
Pattern: ..L.I(S)
Pattern Probability: 0.0002053
Present in taxon: Eukaryota
o See 5 Instances for LIG_IRF3_LxIS_1
o Abstract
Sensing of pathogenic microbes and tissue damage by the innate immune system triggers immune cells to secrete cytokines that promote host defence. Type I IFNs are the key cytokines mediating innate antiviral immunity (Stetson,2006). ViralRNA, cytosolic DNA, and the bacterial cell wall component lipopolysaccharide activate defensive signalling pathways through a number of pattern recognition receptor (PRR)–adaptor protein pairs, including RIG-I–MAVS, cGAS-STING, andTLR3/4-TRIF (Liu,2015). Microbial double-stranded (ds) DNA in the cytosol is sensed by cGAS which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING that is located at the endoplasmic reticulum (ER) surface. Viral dsRNA is sensed in the cytosol by Rig-I-like receptors (RLRs) which activate the adaptor protein MAVS that is located at the mitochondrial surface. Membrane anchored toll-like receptors (TLRs) 3 and 4, which recognize viral dsRNA and bacterial LPS, respectively, when activated recruit the adaptor protein TRIF. The three adaptor proteins STING, MAVS and TRIF contain a conserved motif previously referred as pLxIS that is phosphorylated by TBK1 or IKKepsilon (Liu,2015).
Once phosphorylated, these three adaptor proteins bind to the transcription factor interferon regulatory factor 3 (IRF-3) with affinities between 43 and 104 μM, resulting in TBK1-dependent phosphorylation of an additional pLxIS motif in IRF-3. Phosphorylated IRF-3 subsequently dissociates from the adaptor protein and dimerizes through the same phospho-binding domain that activates the protein (Zhao,2016). The IRF-3 dimer translocates to the nucleus and positively regulates the transcription of IFN-beta (Honda,2006).
The key interaction is between the pLxIS motifs and the IRF-3 C-terminal region that harbours the positively charged surfaces. But other interactions such as the electrostatic and hydrophobic interactions upstream of the motif are also necessary for effective binding.

Interestingly, the rotavirus E3 ubiquitin ligase non-structural protein 1 (NSP1) also contains the pLxIS motif which binds to the same binding region in IRF-3 preventing its activation and promoting its degradation. Phosphorylation of the Ser is not necessary for a low affinity binding with IRF-3 (~200 μM), however, a competitive affinity with respect to the other adaptor proteins (16 μM) is observed when the Ser is phosphorylated. Thus, rotavirus is able to escape innate immune recognition by interfering with the IRF-3-dependent pathway (Barro,2005).
o 5 selected references:

o 25 GO-Terms:

o 5 Instances for LIG_IRF3_LxIS_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
205 210 GSPASLASNLEISQSPTMPF TP 6 Homo sapiens (Human) [new]
Q86WV6 TMEM173
361 366 TSTMSQEPELLISGMEKPLP TP 7 Homo sapiens (Human) [new]
437 442 SPFSGCFEDLAISASTSLGM TP 7 Homo sapiens (Human) [new]
Q99FX5 Non-structural protein 1
484 489 SGTLTEEFELLISNSEDDNE TP 4 Simian rotavirus A/SA11-4F [new]
Q14653 IRF3
391 396 ASSLENTVDLHISNSHPLSL TP 5 Homo sapiens (Human) [new]
Please cite: ELM 2016-data update and new functionality of the eukaryotic linear motif resource. (PMID:26615199)

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