The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
IRF interaction and dimerisation motifs
Functional site description:
The recognition of pathogen-associated molecular patterns (PAMPs) involves different pathways that can trigger convergent immune responses. Following microbial and viral infections, various innate adaptor proteins like STING, MAVS, and TICAM1 relay signals downstream and trigger the activation of IRF transcription factors to produce type I interferons (IFNs) that are essential for host protection. IRF family members such as IRF-3 and IRF-7 are activated by binding to a conserved pLxIS/pLxLS motif that is present in adaptor proteins phosphorylated by TBK1 or IKKε. The phosphorylated motif can bind IRFs, resulting in TBK1-dependent phosphorylation of an additional motif instance present in IRFs. Phosphorylated IRFs can form homo- or hetero-dimers that activate the protein and positively regulate the transcription of IFN-β. The rotavirus NSP1 protein also contains a pLxIS motif which binds to the same region in IRFs thus escaping innate immune recognition by interfering with IRF-dependent pathways.
ELMs with same func. site: LIG_IRF7_LxLS_2  LIG_IRFs_LxIS_1 
ELM Description:
IRF-3, -5 and -6 all utilise the pLxIS motif. Upon infection, the Ser/Thr kinases TBK1 and Ikkε phosphorylate IRF-3 on its pLxIS motif at the C-terminal autoinhibitory region. Phosphorylation triggers the unfolding of this region and induces the homo- or hetero-dimerization of IRF-3, mediated by the pLxIS motif with the subsequent exposure of a binding interface for CREB binding protein (CBP) or p300 on the other side of the same domain (7JFL) (Qin,2005). Once the dimer is translocated to the nucleus, IRF-3 associates with CBP to promote gene binding and activation of IFN-β (Sato,1998). The pLxIS motif and a key upstream phosphoserine is observed in Homo sapiens and also conserved in mice (7JFM), although the mechanism of complex formation between IRF-3 and CBP in these species are similar but somewhat distinct (Jing,2020).

IRF-3 activation is initiated when it binds to innate adaptor proteins like STING, MAVS or TRIF through their conserved phosphorylated pLxIS motif. The pLxIS motif of the adaptor proteins binds to IRF-3 similarly to how the IRF-3 motif binds in its autoinhibited or dimer states, but residues upstream of the motif confer specificity to the different binding partners (Zhao,2016).

In the pLxIS motif, p represents a hydrophilic residue, which is followed by two or three hydrophobic residues (including L and I) and S, which represents the phosphorylated serine. A hydrophobic residue engaged in motif binding is also conserved 1 to 3 residues upstream of the core motif but was not included in the original description. IRF-3 itself contains the pLxIS motif where it is important for homo- and hetero-dimerization, and activation.

A motif mimic is found in rotavirus NSP1 protein which can bind weakly in the unphosphorylated state, and more strongly after phosphorylation (Zhao,2016). All motif instances interact with the same ligand-binding surface of IRF-3. NSP1 antagonizes IRF5 too, according to cotransfection analysis (Barro,2007).
Pattern: [VILPFYM].{1,3}L.I(S)
Pattern Probability: 0.0001795
Present in taxons: Eukaryota Viruses
Interaction Domains:
o See 9 Instances for LIG_IRFs_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 Interferons (IFN), including IFN-α and IFN-β, are major cytokines secreted when immune cells sense pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors (PRRs) (Stetson,2006). Cytosolic PRRs include RIG-I-like receptors, which detect viral dsRNA and are then recruited to the mitochondrial surface where they associate with the mitochondrial antiviral-signalling protein (MAVS). Another cytosolic PRR is the cyclic-GMP-AMP synthase (cGAS) which, in response to the presence of viral DNA, catalyses the production of cGAMP. This second messenger leads to the activation of the endoplasmic reticulum membrane protein stimulator of IFN genes (STING) (Sun,2013). Toll-like receptors (TLRs) are membrane-bound PRRs that recognize different types of bacterial, viral or fungal PAMPs. For example, TLR3 responds to viral dsRNA, while TLR4 recognizes lipopolysaccharides from gram-negative cell bacteria (Akira,2006). TLRs recruit adaptor proteins such as the TIR-domain-containing adaptor protein (TRIF, also called TICAM1) to further induce the interferon response pathway. The phosphorylation of the adaptor proteins MAVS, cGAS and STING is essential for downstream signalling. The three proteins share a conserved linear motif referred to as pLxIS that is phosphorylated by TANK-binding kinase 1 (TBK1) and/or IκB kinase-ε (IKKε) (Liu,2015). Once phosphorylated and helped by other electrostatic interactions and a semi-conserved hydrophobic residue 1 to 3 positions upstream, the motif binds to positively charged surfaces of IRF-3, IRF-5, IRF-7 (Lazear,2013) and possibly some other Interferon Regulatory Factor paralogues (IRFs).

The IRF family consists of nine transcription factors (IRF1-9) with different defence roles (Savitsky,2010). Among them, IRF-3 and IRF-7 are engaged in innate immunity against bacteria and viruses as the key IRFs for IFN production (Wu,2014). While they are similar in sequence and structure and tend to form heterodimers (Schmid,2014), IRF-3 is expressed ubiquitously while IRF-7 is generally present at low concentrations (Au,1998) but responds to a feedforward loop induced by IFNs (Marie,1998). IRF-5 and IRF-6 are more structurally related, with the former linked to pro-inflammatory response and the regulation of apoptosis, and the latter involved in the regulation of cell proliferation and the differentiation of keratinocytes (Taniguchi,2001).

Regardless of their heterogeneity, the signal response (or serine-rich) domain (SRD) adjacent to the C-terminal IRF-Associated Domain (IAD) of IRF-3, IRF-5, IRF-6 and IRF-7 (PF10401) carry variants of the same pLxIS motif that can also be phosphorylated by TBK1/IKKε. Phosphorylation of these IRFs promote their dissociation from the adaptor proteins and subsequent homo- or hetero-dimerization through the pLxIS motifs, which bind the same phosphopeptide-binding domain that is then blocked for further interaction with adaptor proteins and enabling transition into the nucleus (Zhao,2016). For example, the C-terminal region of IRF-3 undergoes opening following phosphorylation, with the subsequent exposure of a binding interface for CREB binding protein (CBP) or p300 (7JFL; Qin,2005) allowing activation of innate immunity gene expression. Likewise, IRF-5 is activated by phosphorylation by IKKβ at conserved residues of the C-terminal domain. This also releases autoinhibition by a conformational rearrangement, followed by translocation of IRF-5 to the nucleus and interaction with CBP (Ren,2014). The crystallization of IRF-5 with phosphomimetic mutations reveals an elongated structure in the C-terminal region (3DSH). This conformation promotes dimerization and positions the binding interfaces to interact with the CBP/p300 dimers. However, the affinity is 1-2 orders of magnitude lower compared to IRF-3, which is expected given the relatively short length of the peptide assayed, but could nevertheless indicate a finely tuned switch between autoinhibition and dimerization in IRF-5. This difference in affinity may highlight distinct functional requirements: IRF-3 is expressed constitutively to offer a highly sensitive response to viral infection, while IRF5 functions as a more stable dimer with greater basal activity (Chen,2008). Structurally similar to IRF-5 and with a very similar pLxIS motif variant, IRF-6 has a different functional role which is not conserved in vertebrates. In mammals, IRF-6 has a developmental role not related with IFN induction (Kawasaki,2016). But it has been suggested that IRF-6 is a positive modulator of IFN expression in zebrafish, including the activation of the transcription of MAVS, RIG-I and other host genes (15939375). The pLxIS motif is needed for IRF-6 dimerization, a key step for its activation and nuclear translocation, as demonstrated by coimmunoprecipitation of phosphomimetic double serine mutants (Kwa,2014). The motif in IRF-7 is a variant, pLxLS, with Leu for the usual Ile (Lin,2000).

It is not documented that other IRF family members interact with the pLxIS motif. The IAD domain that is conserved in IRF-1 and IRF-2 differs from that of other IRFs (Yanai,2012). IRF-4 and IRF-8 have a central role in controlling the development of dendritic cells and thus are prone to interact with other partners (PMD:30984161). IRF-9 does not show autoinhibition, and the structural evidence of IRF-9 in complex with STAT2 (5OEN) shows a specific IAD interaction interface that does not involve a linear motif (Rengachari,2018).

Interestingly, the rotavirus non-structural protein 1 (NSP1) also contains the pLxIS motif which binds to the same phosphopeptide-binding region of IRFs. The interaction prevents the activation of IRF-3, IRF-5 or IRF-7 by phosphorylation, promoting their degradation instead. Phosphorylation of the Ser is not necessary for a low affinity binding (e.g. ~200 μM with IRF-3), but favours a more competitive affinity with respect to the host's other adaptor proteins (16 μM for IRF-3, against 43 to 104 μM measured for cellular adaptor proteins) (Zhao,2016). Upon binding, NSP1 can behave as an E3 ubiquitin ligase, directing IRFs with IAD regions into the ubiquitin proteasome system (Arnold,2013). In this way, rotavirus is able to escape innate immune recognition by interfering with the IRF-dependent pathways (Barro,2005). This raises the possibility that other pathogens may mimic the pLxIS motif to disrupt innate immunity.
o 7 selected references:

o 25 GO-Terms:

o 9 Instances for LIG_IRFs_LxIS_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
O14896 IRF6
419 424 RSFDSGSVRLQISTPDIKDN TP 2 Homo sapiens (Human)
Q13568 IRF5
441 446 LSWSADSIRLQISNPDLKDR TP 5 Homo sapiens (Human)
Q14653 IRF3
391 396 ASSLENTVDLHISNSHPLSL TP 5 Homo sapiens (Human)
Q99FX5 Non-structural protein 1
484 489 SGTLTEEFELLISNSEDDNE TP 4 Simian rotavirus A/SA11-4F
Q86WV6 TMEM173
361 366 TSTMSQEPELLISGMEKPLP TP 7 Homo sapiens (Human)
P70671 Irf3
383 388 GASSLKTVDLHISNSQPISL TP 1 Mus musculus (House mouse)
Q3TBT3 Tmem173
360 365 PSVLSQEPRLLISGMDQPLP TP 2 Mus musculus (House mouse)
203 210 TGSPASLASNLEISQSPTMP TP 6 Homo sapiens (Human)
436 442 SPFSGCFEDLAISASTSLGM TP 7 Homo sapiens (Human)
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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