Review

Immunology and Cell Biology (2007) 85, 411–419; doi:10.1038/sj.icb.7100095; published online 31 July 2007

Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins

Tanya M Watters1, Elaine F Kenny1 and Luke A J O'Neill1

1School of Biochemistry and Immunology, Trinity College, Dublin, Ireland

Correspondence: TM Watters, School of Biochemistry and Immunology, Trinity College, Dublin, Ireland. E-mail: wattert@tcd.ie

Received 13 June 2007; Accepted 15 June 2007; Published online 31 July 2007.

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Abstract

The Toll/IL-1 receptor (TIR) domain plays a central role in Toll-like receptor (TLR) signalling. All TLRs contain a cytoplasmic TIR domain, which, upon activation, acts as a scaffold to recruit adaptor proteins. The adaptor proteins MyD88, Mal, TRIF, TRAM and SARM are also characterized by the presence of a TIR domain. MyD88, Mal, TRIF and TRAM associate with the TLRs via homophilic TIR domain interactions whereas SARM utilizes its TIR domain to negatively regulate TRIF. It is well established that the differential recruitment of adaptors to TLRs provides a significant amount of specificity to the TLR-signalling pathways. Despite this, the TIR–TIR interface has not been well defined. However, structural studies have indicated the importance of TIR domain surfaces in mediating specific TIR–TIR interactions. Furthermore, recent findings regarding the regulation of adaptors provide further insight into the crucial role of the TIR domain in TLR signalling.

Keywords:

toll-like receptors, signal transduction, TLR adaptors, TIR domain, innate immunity, inflammation

The innate immune system is conserved among vertebrates and represents the first line of defence against invading microbial pathogens.1 Originally, it was thought to function largely in a non-specific manner. This view, however, was challenged by Janeway in 1989,2 who postulated that the innate immune system specifically detects pathogens via germ line-encoded receptors termed pattern recognition receptors that recognize highly conserved microbial structures and thereby initiate an immune response. The first proof of specificity in innate immunity came with the realization that the Drosophila protein Toll (dToll), which had initially been identified as having a role in embryonic dorso-ventral patterning, was critical for an effective immune response to fungus Aspergillus fumigatus in the adult fly.3 Soon thereafter, a human homologue of Toll (hToll, later called Toll-like receptor 4, TLR4) was discovered, which had the ability to induce the production of inflammatory cytokines when constitutively active.4 Subsequently, mice with a dominant negative mutation in the gene encoding TLR4 showed defective responses to lipopolysaccharide (LPS),5 indicating that TLR4 represented an innate sensor of this bacterial component.

To date, 13 mammalian TLRs have been identified (10 in humans and 12 in mice).6 The TLRs are type 1 transmembrane receptors which contain an N-terminal, leucine-rich repeat (LRR) domain, a transmembrane region and a C-terminal cytoplasmic domain.7 The cytoplasmic domain shares a high degree of homology with that of the type-1 IL-1 receptor (IL-1R2) and is, therefore, termed the Toll/IL-1R (TIR) domain.8 TLRs are differentially expressed on a wide range of cell types with haematopoietically derived cells such as macrophages and dendritic cells expressing almost a full repertoire.7 While the TLRs that are mainly responsible for the detection of bacterial products (TLRs 1,2 ,4 ,5,and 6) are expressed on the cell surface, the subset of TLRs that sense viral components (TLRs 3, 7, 8 and 9) are located intracellularly on endosomal membranes.9

Upon ligation of TLRs with their cognate ligand, signalling cascades are activated which result in the production of innate effector responses as well as the initiation of an adaptive immune response.10 Adaptor proteins are first recruited to the cytoplasmic domains of the receptors via TIR–TIR interactions. The five adaptors are MyD88, MyD88-adaptor like (Mal, also known as TIRAP), TIR-domain-containing adaptor protein inducing IFN-beta (TRIF, also known as TICAM1), TRIF-related adaptor molecule (TRAM, also known as TICAM2) and sterile alpha- and armadillo-motif containing protein (SARM). MyD88 is utilized by all of the TLRs except TLR3 whereas TRIF signals downstream of TLR3 and TLR4. TLR4 differs from the other TLRs in that it recruits MyD88 and TRIF to its cytoplasmic domain via the bridging adaptors, Mal and TRAM, respectively. TLR2 also utilizes Mal to recruit MyD88. SARM, in contrast to the other adaptors, was found to be a negative regulator of TLR signalling. These adaptors (with the exception of SARM) recruit downstream signalling molecules which lead to the activation of NF-kappaB and members of the IRF family of transcription factors. This ultimately results in the production of pro-inflammatory cytokines and type-1 interferons.11 It has become apparent that these signalling pathways, which play such a crucial role in host defence, can also cause significant immunopathology if overactivated or insufficiently controlled.12 Likewise, a defect in TLR signalling can result in severe immunodeficiency.13

Although we now have a good understanding of the pathways initiated by the TLRs, the picture is still far from complete. Many unanswered questions remain regarding specificity and regulation of the pathways as well as cross talk with other networks. Recently, there have been many detailed reviews on the aspects of TLR signalling. Here, in order to avoid repetition, we focus on the important role of the adaptor proteins in providing specificity in both the initiation and termination of TLR signalling. We discuss in detail our current knowledge of the interface between TIR domains since TLR signalling is initiated via TIR–TIR interactions. We also discuss the various ways in which adaptors are known to be regulated.

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Ligand recognition by TLRs

Upon ligand recognition, TLRs are thought to form homo- or heterodimers or indeed higher oligomers, and this oligomerization is necessary for signalling to occur. 14, 15, 16, 17 TLR2 is known to form heterodimers with TLR1 or TLR6 while TLR4 homodimerizes in a similar fashion to dToll.16, 17, 18 While the TLR2/TLR6 heterodimer detects diacylated lipoproteins from bacteria, the TLR2/TLR1 heterodimer senses triacylated lipoproteins.19 There are also a number of ligands such as lipoteichoic acid and mycobacterial lipoarabinomannan that appear to be recognized by TLR2 independent of TLR1 or TLR6.20 TLR3 detects the double-stranded RNA (dsRNA) from viruses. The ligand for TLR4 is LPS from the cell wall of gram-negative bacteria. TLR5 senses flagellin, a highly conserved component of bacterial flagella. Both TLR7 and TLR8 recognize the single-stranded RNA (ssRNA) from viruses, while TLR9 is responsible for detecting bacterial and viral CpG DNA motifs. In addition, TLRs can be activated by endogenous ligands such as heatshock proteins, hyaluronate and single-stranded RNA, the detection of which may contribute to the development of autoimmune disorders.21 (For a list of TLR ligands, see Table 1).


TLRs have the capability to recognize a diverse range of conserved microbial structures via their extracellular region which is composed of 19–25 tandem copies of the LRR motif. Part of this diversity is thought to be due to insertions at positions 10 and 15 of the LRR consensus sequence.22 Another mechanism whereby the diversity of ligand recognition may be increased is by the use of certain accessory proteins. It seems that an LPS response requires the sequential interaction and transfer of LPS to LPS-binding protein, the glycosylphosphatidylinositol-anchored protein CD14 and MD-2, and the simultaneous engagement of LPS and TLR4 by MD-2.23, 24 It has been shown that the lipid A precursor lipid IVa acts as an agonist for mouse TLR4–MD2 but is antagonistic on chimeric mouse TLR4–human MD-2, which indicates that MD-2 determines the antagonistic activity of lipid IVa, pointing to a role for MD-2 in the specificity of ligand recognition.25 CD14 has also been shown to be required for signalling induced by TLR2 ligands,20 and it has been found recently that the scavenger receptor CD36 is involved in recognition of diacylated lipopeptides by TLR2/6 heterodimers.26 Finally, heterodimerization of TLRs as in the case of TLR2 with TLR1 or TLR6 also increases the repertoire of ligands which can be detected.16

Upon ligand-induced dimerization or oligomerization of the TLRs, it is thought that a conformational change occurs in the cytoplasmic TIR domains, thereby allowing the recruitment of adaptor proteins to the signalling complex27 and subsequent recruitment of downstream signalling molecules. The resulting signalling pathways may generally be grouped into MyD88-dependent and MyD88-independent/TRIF-dependent pathways. (Figure 1 provides an overview of these pathways).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The three signalling pathways. There are three common pathways involved in Toll-like receptor (TLR) signalling. (a) The MyD88-dependent pathway: This pathway is used by all TLRs except TLR3. It is initiated in the case of TLR4 by the TIR domain of Mal binding the TIR domain of TLR4, which has dimerized after ligand binding. Mal recruits MyD88, which binds IRAK4 and -1. IRAK1 is phosphorylated by itself and IRAK4 and leaves the membrane to activate TRAF6. After TRAF6 is ubiquitinated, it interacts with TAB2 to activate TAK1. TAK1 activates the IKK complex and IkappaB is phosphorylated, ubiquitinated and degraded allowing NF-kappaB to translocate to the nucleus to produce proinflammatory cytokines. TAK1 also activates MKK6 which in turn activates JNK and p38 leading to AP-1 activation and the production of proinflammatory cytokines. TRAF6 can also activate IRF5. (b) The TRIF-dependent pathway: This pathway is used by TLR3 and with TLR4 by binding TRAM at the membrane. This pathway begins with the activation of TBK-1 leading to the activation of IRF3, a transcription factor that translocates to the nucleus to produce IFN-inducible genes. Alternatively RIP1 is activated by TRIF and this feeds into the MyD88 pathway by activating TRAF6. (c) The alternative MyD88 pathway: This only occurs in plasmacytoid dendritic cells with the activation of TLR7 and -9 leading to the activation of TRAF6 through MyD88, IRAK4 and -1. This results in the activation of IRF7 which translocates to the nucleus to produce IFN-alpha and IFN-inducible genes.

Full figure and legend (212K)

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MyD88-dependent signalling

As depicted in Figure 2, MyD88 has a modular structure, comprising a C-terminal TIR domain and an N-terminal death domain (DD), separated by a small intermediate domain (ID).28 MyD88 was first shown to play a role as an adaptor in IL-1 signalling, serving to couple the IL-1R to signalling intermediates.29, 30 Subsequently, it was demonstrated that MyD88 served an analogous function downstream of TLR4.31 MyD88-deficient mice lose the ability to produce pro-inflammatory cytokines in response to a large range of TLR ligands revealing the crucial role of this protein in TLR signalling.32, 33, 34 MyD88 is recruited to the cytoplasmic portion of the TLR and interacts with IRAK4 and IRAK1 via homophilic DD interactions. Activated IRAK4 phosphorylates and activates IRAK1 which subsequently interacts with TNFR-associated factor-6 (TRAF6),35 causing the oligomerization and activation of TRAF6.30, 36, 37, 38, 39 TRAF6 is a ubiquitin E3 ligase and functions with the ubiquitin-conjugating enzyme Ubc13 and the Ubc-like protein Uev1a to catalyze the synthesis of Lys63-linked polyubiquitin chains on target proteins including TRAF6 itself.40, 41 Ubiquitinated TRAF6 then recruits TAB2 and activates the TAB2-associated TAK1 kinase.40, 41, 42 Activated TAK1 then activates the IKK complex which phosphorylates IkappaB resulting in its ubiquitination and subsequent proteosome-mediated degradation. This leaves NF-kappaB free to translocate to the nucleus and initiate gene transcription.43, 44 TAK1 is also responsible for the activation of MKK6 which phosphorylates and activates the kinases JNK and P38 and thereby mediates AP-1 activation.44

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Domains of the adaptor proteins. There are five adaptors known to date. MyD88 is 296 amino acids in length and contains two domains. At the C terminus (aa 1–110) is the death domain (DD) and at the N terminus (aa 155–296) is the TIR domain. Mal is 256 amino acids long and contains many domains and phosphorylation sites. At the N terminus, there is a PIP2-binding domain (aa 15–35). This is followed by the TIR domain (aa 86–188) and a TRAF6 domain (aa 188–196). There are two phosphorylation sites for Bruton's tyrosine kinase at positions 86 and 187. Located at position 180 is the serine/leucine site linked to the genetic susceptibility to several diseases including TB and malaria. Finally, at position 198 is the aspartic acid indicating the presence of the caspase-1 cleavage site. TRIF is the longest adaptor at 712 amino acids. This consists of a TRAF6-binding domain (aa 230–235), the TIR domain (aa 380–530) and a receptor-interacting protein (RIP) homotypic interaction motif (RHIM) (aa 661–699). TRAM, the smallest at 235 amino acid long, contains a myristoylation site at its N terminus followed by serine at position 16 which is phosphorylated by protein kinase C-alt epsilon. Its TIR domain is located between amino acids 75 and 235. SARM is 690 amino acids in length and is made up of several domains all located near the N terminus. There are two SAM motifs between amino acid 375 and 515 followed by the TIR domain between 515 and 660.

Full figure and legend (175K)

MyD88 also participates in the activation of several members of the IRF family of transcription factors, namely, IRF1, IRF7 and IRF5. MyD88 is necessary for the activation of IRF7 downstream of TLR 7, 8 and 9 in plasmacytoid dendritic cells (pDCs). In turn, IRF7 activation leads to the production of IFNalpha.45, 46, 47 Upon ligand binding to TLR 7, 8 or 9, IRF7 is recruited to a complex containing MyD88, TRAF6, IRAK4 and IRAK1.47, 48, 49 Phosphorylation of IRF7 by IRAK1 in this complex was found to be essential for its transcriptional activation. It appears that IRAK4 plays a role in IRF7 activation solely by phosphorylating and activating IRAK1.49 A recent study demonstrated that IRF5 is activated by the TLR–MyD88 pathway and has an important role in the induction of pro-inflammatory cytokines. In cells from IRF5-deficient mice, the induction of inflammatory cytokines is severely impaired, whereas IFN-alpha production is not affected. It was shown that IRF5 interacts with, and is activated by MyD88 and TRAF6 causing the nuclear translocation of IRF5 and the expression of inflammatory cytokines.50 Recent evidence also identified IRF1 as an additional transcription factor activated by MyD88. Upon TLR activation, IRF1 is activated by MyD88, causing it to translocate to the nucleus where it induces a specific gene subset including IFN-beta, inducible NO synthase and IL-12p35.51

Mal was the second TIR domain-containing adaptor to be identified.52, 53 Studies utilizing Mal-deficient mice revealed that it lay on the MyD88-dependent pathway and functioned as a bridging adaptor for MyD88; recruiting it to the cytoplasmic domain of TLR4 and TLR2.54, 55 Mal-deficient mice were found to have an identical phenotype to MyD88-deficient mice, but only in terms of responsiveness to TLR4 and TLR2 ligands. Activation of NF-kappaB and MAPKs by TLR4 was induced with delayed kinetics in cells from these mice, and they also showed an impaired production of cytokines such as TNF and IL-6. Furthermore, signalling by TLR2 was abolished in Mal-deficient cells.54, 55

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MyD88-independent/TRIF-dependent signalling

The fact that MyD88-deficient cells retained the ability to activate NF-kappaB and MAPKs in response to LPS and Poly I:C32 suggested the existence of a MyD88-independent pathway. This led to the identification of the adaptor TRIF, which activates IRF3 and NF-kappaB downstream of TLR3 and TLR4 in a MyD88-independent manner.55, 56, 57 TRIF-deficient mice were found to be defective in IRF3 activation and the production of IFN-beta mediated by TLR3 and TLR4, and inflammatory cytokine production was also impaired in response to LPS.57 TRIF mediates IRF3 activation via TRAF family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1), which associates with the N terminus of TRIF.58, 59 Recently, both TRAF3 and NAK-associated protein 1 (NAP1) have been implicated in mediating the activation of TBK1 by TRIF by acting as a bridge between the two proteins.60, 61, 62 It has been reported that TRAF6 associates with the N terminus of TRIF and is required for TRIF-mediated NF-kappaB activation.59, 63 However, NF-kappaB activation was not affected downstream of TLR3 in macrophages from TRAF6-deficient mice and delayed NF-kappaB activation was still observed in response to TLR4 ligands, suggesting that TRAF6 is not involved in TRIF-dependent NF-kappaB production.64 TRIF also associates with receptor-interacting protein 1 (RIP1) via its C-terminal RIP homotypic interaction motif (RHIM), and in the absence of RIP1, TLR3-mediated NF-kappaB activation was abolished, suggesting that RIP1 mediates the TRIF-induced NF-kappaB activation.65 Additionally, LPS failed to stimulate NF-kappaB activation in RIP1/MyD88 double deficient cells which reveals that RIP1 is also required for the TRIF-dependent TLR4-induced NF-kappaB pathway.66

TRAM is utilized solely by TLR4. It functions as a bridging adaptor for the MyD88-independent signalling pathway, serving to recruit TRIF to TLR4.67 TLR4- but not TLR3-mediated MyD88-independent IFN-beta production and activation of signalling cascades were abolished in TRAM-deficient cells.68

SARM, the fifth adaptor TIR-domain containing adaptor to be discovered, is distinct from the other adaptors in that it does not activate NF-kappaB or IRF3,68 but instead, acts as a negative regulator of TLR signalling.69 In addition to a C-terminal TIR domain, SARM contains two tandem sterile alpha-motif (SAM) domains and has also been predicted to contain heat-armadillo motifs. SARM was shown to specifically target TRIF. SARM associates with TRIF and blocks TRIF-induced NF-kappaB activation. Furthermore, silencing of SARM expression by small interfering RNA resulted in an enhancement of TRIF-dependent signalling. The TIR domain and two SAM domains of SARM are necessary for inhibition of signalling and the N terminus is required for LPS-induced enhancement of SARM expression. The exact mechanism whereby SARM inhibits TRIF signalling has not been elucidated but it is likely that SARM acts to sequester TRIF, and prevent its interaction with downstream effectors. Another possibility is that SARM interacts with TRIF via the TIR domain and recruits an inhibitory protein via the SAM domains.69

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Initiation of signalling

It would seem that for different adaptors to be recruited to various TLRs, there must be quite a degree of specificity in the TIR domain interactions that occur. Indeed, the structural evidence obtained to date suggests that the surface of the TIR domain is largely responsible for this specificity. Not only are these TIR–TIR interactions crucial for signalling initiation, they also confer a high degree of specificity to the signalling pathways.

It is generally accepted that similar to other type 1 transmembrane receptors, TLRs require ligand-induced dimerization or crosslinking to establish intracellular signal transduction.17, 70 In a study that examined the interaction of dToll with its ligand Spaetzle, it was concluded that Spaetzle binds directly to the Toll ectodomain, thereby crosslinking two receptors, and this dimerization was necessary for signalling to occur.17 It was also suggested that binding of dsRNA to the extra-cellular domain of TLR3 promotes multimerization of the receptor.14, 15 Contacts between both the extracellular and intercellular domains are involved in receptor dimerization.66 It has been suggested that TLRs exist as non-functional dimers in the absence of ligand and that ligand binding induces conformational changes which allow stable receptor association.27 Ligand binding is therefore likely to lead to a conformational rearrangement of the cytoplasmic domains, creating a scaffold to which TIR domain containing adaptors can be recruited.37

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Structure of the TIR domain

The TIR domain is roughly 200 residues in length and contains three highly conserved regions, denoted box 1, box 2 and box 3.8, 71 Mutagenesis studies involving the IL-1R TIR domain showed that box 1 and box 2 are essential for signalling. Reports also indicate that box 3 is involved in signalling72, 73 although it may not have as important a role as box 1 and box 2.74

Information regarding the TIR domain structure is quite limited; only the crystal structures of the TLR1 and TLR2 TIR domains have been obtained to date75 and subsequent studies have used these structures as a template to create models of other TIR domains. The structure of the TIR domain is similar to that of the bacterial chemotaxis protein CheY,76, 77 consisting of a central five-stranded parallel beta-sheet (betaA–betaE) surrounded by five helices (alphaA–alphaE). The loops are named according to the secondary structural elements they connect (For example, the BB loop connects strand betaB and helix alphaB).75 Most of the conserved residues in the TIR domain are found buried in the core of the fold.76 The study on the TIR domains of TLR1 and -2 as well as the observation that the sequence conservation among TIR domains in general is 20–30% suggests that there are significant structural differences among various TIR domains which would contribute to specificity in the signal transduction process.75 In general, the loop regions are the most variable in sequence and structure, often containing large insertions or deletions, and several of these regions have been implicated in the TIR–TIR interactions.39, 75, 76, 78, 79, 80, 81 However, the loops also contain certain conserved residues which may be important for making contact with other TIR domains.

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Role of the BB loop

Considering the importance of TIR domain interactions in the initiation of signal transduction, structural knowledge of these interactions is relatively sparse. Despite this, however, mutagenesis and structural studies that have been carried out to date are helping us to build a clearer image of the TIR–TIR interface. It seems as though various loop regions of the TIR domains play an important role in mediating the specificity of TIR domain interactions. The most extensively studied are the BB and DD loops.

In particular, much attention has been focused on the BB loop as a structural determinant of specificity. The BB loop comprises part of box 2 and contains a number of highly conserved residues. The crystal structure of the TIR domain indicates that the BB loop forms a protrusion which extends from the surface of the TIR domain.75 Notably, in most TIR domains, the BB loop contains an invariant proline which is important for signalling. In CH3/HeJ mice, mutating this proline to a histidine in TLR4 (TLR4 P712 H) results in hyporesponsiveness of these mice to LPS.5 The equivalent mutation in TLR2 (TLR2 P681 H) prevents signalling by the receptor75, 80 and TLR2 P681H acts as a dominant negative inhibitor of TLR2-mediated activation of NF-kappaB.16 Also, a peptidomimetic based on the BB loop of MyD88 abolishes signalling by the IL-1R and prevents the association of MyD88 with the IL-1R accessory protein (IL-1RAcP).82 The crystal structure of the TIR domain of the TLR2 P681 H mutant did not show any significant structural changes caused by the mutation. It was, therefore, suggested that this mutation abolishes signalling by disrupting a direct point of contact between TIR domains rather than disrupting the structure of the domain.75 It was reported that the P681H mutation in TLR2 prevented the interaction of the receptor with MyD88.75, 83 In contrast to these findings, however, the TLR4 P712H mutation did not affect the association of TLR4 with MyD8876, 82 or Mal.76 Nor did the equivalent mutations in MyD88 or Mal, prevent the binding of these adaptors to TLR4 or their hetero- or homodimerization.76 It is likely that other residues in the BB loop apart from the conserved proline are involved in TIR domain interactions. For example, mutations at almost every position in the BB loop of TLR4 or dToll leads to significantly reduced signalling activity of the receptors. Also, when glycine 676 in the BB loop of the TLR1 TIR domain was mutated to leucine, signalling was abrogated, possibly because of the inability of the mutant TLR 1 to dimerize with TLR2.78 However, the role of the BB loop in TIR domain interactions is still unclear.

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The TIR–TIR interface

Basically, three TIR–TIR interfaces can be identified with respect to TLR signalling: the receptor–receptor, receptor–adaptor and adaptor–adaptor interfaces. A number of different models for TIR-domain interactions have been proposed based on the crystal structures of TLR1 and -2.

Dunne et al.76 modelled the TIR domains of TLR4, MyD88 and Mal. This study indicated that there are non-overlapping binding sites on TLR4 for MyD88 and Mal. It was predicted that Mal associated with TLR4 through its DD and DE loops with a region of TLR4 adjacent to its BB loop. MyD88 was predicted to bind via its AA and DD loops to the CD loop on TLR4 which, interestingly, is located on the surface of the receptor, opposite to BB loop. In contrast, docking studies with TLR2 indicated that MyD88 and Mal bind to the same surface of TLR2 with the BB loop of TLR2 forming a possible point of contact. It was also suggested that MyD88 and Mal would interact with one another at a third overlapping site with the BB loop and the fourth alpha-helix making significant contributions. The electrostatic surfaces of TIR domains were found to be quite distinct which suggests that electrostatic complimentarity may also play a role in adaptor recruitment.

Tao et al.80 observed the formation of an asymmetric dimer in the crystal structure of TLR2 in which the DD loop of one molecule and the BB loop of the second molecule contribute to contacts at the interface. The authors postulate that the asymmetric dimer may reflect the natural heterodimeric TLR2/TLR1 or TLR2/TLR6 complexes with the DD loop of TLR2 and the BB loop of TLR1/6 involved in dimerization. The same conclusion was reached by Gautam et al.78 Here, a random mutagenesis analysis of the TLR2 TIR domain was carried out and the resulting mutants were screened for deficiencies in TLR1/2 signalling. With the aid of docking studies it was shown that residues Arg-748 and Phe-749 in the DD loop of TLR2 were involved in close contact with Gly-676 in the BB loop of TLR1. This would theoretically leave the BB loop of TLR2 available for recruitment of adaptor molecules.

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The Poc site

In a recent study using a forward genetic technique, Jiang et al.79 discovered another site which is important for TIR domain interactions. The phenovariant, Pococurante, or Poc was attributed to a point mutation, I179N, in the TIR domain of MyD88. Poc was found to abolish NF-kappaB activation downstream of TLRs in response to most ligands. Interestingly the Poc mutation in TLR2 permitted sensing of diacyl lipopeptides (sensed by TLR 2/6 or TLR2 alone) but not triacyl lipopeptides (sensed by TLR 2/1) indicating that TLR2 activates downstream signalling in a manner distinct from other TLRs. Structural analysis predicted that, similar to the BB loop, the Poc site protrudes from the surface of the TIR domain and is surrounded by hydrophobic residues. Based on the computational docking studies, it was postulated that BB loops and Poc sites interact homotypically across the receptor–adaptor signalling interface, whereas the conserved C-terminal alphaE-helices are responsible for adaptor–adaptor and receptor–receptor oligomerization. They also hypothesized that the interaction between the receptor and the adaptor can, in general, be maintained by either the Poc site or the BB loop and that both are needed for signalling to occur, except in the case where TLR2 is stimulated by diacyl lipopeptides. This would provide an explanation for why BB loop mutations can abolish signalling downstream of TLR4, while the interaction of adaptor and receptor is maintained.76

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Remaining issues regarding TIR domain interactions

These apparent contradictory findings may be due to a number of factors. Firstly, most of the structural studies to date have been carried out using models of TIR domains due to the lack of crystal structure availability (with the exception of the TLR1 and -2 TIR domains). Obviously, this is not the ideal as different modelling programs may produce varying results. Secondly, despite having a number of highly conserved residues, there is still a significant amount of sequence diversity among TIR domains and it is predicted that structural diversity is also high.75 It would not be surprising then, that certain results obtained from studying one TIR domain may not hold for all TIR domains. It has been noted that the BB and DD loops are conformationally flexible76, 78 and so it is reasonable to suppose that these regions may undergo conformational changes upon ligand binding to TLR ectodomains,76 or that they may adopt different conformations in different TIR domains. It is also possible that protein sequences outside of the TIR domain may affect the overall secondary structure of the TIR domain which is not apparent when the TIR domain is studied in isolation.

To date, little is known about how the adaptors TRIF and TRAM may be recruited to receptor TIR domains. Blocking peptides (BPs) corresponding to the BB loops of TRIF and TRAM disrupted the activation-signalling pathways in response to LPS.81 However, the molecular basis for this inhibition was not determined. Also, the ability of the TRIF BP to block signalling downstream of TLR3 was not tested. As TLR3 is the only TLR which does not contain the conserved proline in the BB loop, it would be interesting to establish if this would affect the ability of the TRIF BP to abrogate signalling. Even though the exact interfaces between the TIR domains have not yet been established, it is clear that there is a great deal of variation in the way two TIR domains can interact and that this variation is due to the structural differences. The finding by Jiang et al.79 showing that TLR2 differentially engages MyD88 in response to different ligands suggests that ligand binding to different regions of the LRR domain would play a significant role in the ensuing conformational change in the TIR domain. Another possible explanation is that the TIR domain of TLR2 adopts different conformations depending on whether it dimerizes with TLR1 or TLR6.

For a definitive answer to the outstanding questions, it will be necessary to obtain structures of oligomerized receptors, adaptors and adaptor–receptor complexes, and also, structural changes of the TIR domain upon ligand binding need to be addressed. As the crystallization of these structures is technically demanding, we may have to rely largely on models of TIR domains for some time. As modelling analysis is carried out on additional TIR domains, we will gain a greater insight into the inherent difference in various TIR domain interactions.

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Regulation of adaptors

In addition to the specificity involved in the interactions of the adaptor TIR domains with the intracellular regions of the TLRs, the function of the adaptors is regulated by diverse mechanisms which provide an extra level of specificity to the pathways. Some of these mechanisms would be predicted to impinge on the TIR domain in some way, either changing its structure so that the adaptor interacts differently with other proteins, or modifying the availability of the adaptor, and so preventing homophilic TIR interactions.

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MyD88

MyD88 is the central adaptor in inflammatory responses so it is not surprising that there are mechanisms by which MyD88 is negatively regulated; some of which involve TIR domain interactions. Transforming growth factor-beta (TGF-beta) partly exerts its anti-inflammatory effects by causing direct ubiquitination of MyD88, thereby augmenting its degradation by the proteasome. This would, in turn, disrupt the signalling complex at the cytoplasmic domain or the TLR. It was found that the membrane bound form of ST2, which contains a TIR domain, specifically inhibited NF-kappaB activation downstream of IL-1R, TLR2, TLR4 and TLR9 but not TLR3. This involves the sequestration of MyD88 as well as Mal via their TIR domains. This serves as another example of specificity in TIR domain interactions, although this time it is in the context of signal termination. MyD88 short (MyD88 s) is an alternatively spliced form of MyD88 which lacks the ID.36, 38 MyD88 s acts as a dominant-negative inhibitor of IL-1- and LPS-, but not TNF-induced NF-kappaB activation.28 This inhibitory activity is due to the inability of MyD88 s to recruit IRAK4, thereby preventing IRAK4-induced IRAK1 phosphorylation.36

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Mal

As shown in Figure 2, Mal contains a putative TRAF6-binding motif within its TIR domain. Indeed, Mal could associate with TRAF6 in a co-immunoprecipitation assay. Unlike wild-type Mal, Mal E190A, harbouring a mutation within its TRAF6-binding domain, was unable to activate NF-kappaB and inhibited TLR2- and TLR4-mediated activation of NF-kappaB in a dose-dependent manner. This suggests an independent role for Mal in regulating NF-kappaB-dependent gene transcription by using its TIR domain to interact with TRAF6.84

A recent study showed that Mal also contains a C-terminal phosphatidylinositol 4,5-bisphosphate (PIP2)-binding domain distinct from its TIR domain which serves to localize it to the plasma membrane. When this PIP2-binding domain was mutated, Mal was not recruited to the plasma membrane, nor did it have the ability to recruit MyD88, and signalling in response to LPS was abrogated. This confirms the role of Mal in recruiting MyD88 to the membrane. Furthermore, when Mal was localized to portions of the membrane by a PIP2-independent mechanism, signalling through TLR4 could not occur. This suggests that TLR signalling may be controlled by phospholipid metabolism.85, 86

It was also shown that Mal is phosphorylated by Bruton's tyrosine kinase (Btk) on tyrosine residues at positions 86 and 187, and this event is necessary for efficient TLR4 signalling. Notably, Tyr-86 and Tyr-106 are predicted to be located at the surface of the TIR domain, which would leave them accessible for phosphorylation. It is likely that this tyrosine phosphorylation would cause a conformational change in the TIR domain of Mal, thereby regulating its interaction with other proteins.87

Mal, like many components of the TLR-signalling pathway, is subject to negative regulation. A recent study demonstrated that Mal undergoes degradation mediated by SOCS1. Here it was shown that SOCS1 mediates polyubiquitination of Mal on two N-terminal lysine residues, thereby mediating Mal degradation via the 26S proteasome. A prerequisite for Mal degradation is tyrosine phosphorylation of Mal by Btk as this may allow SOCS1 to associate with Mal through its SH2 domain. As already mentioned, phosphorylation of Mal by Btk was found to potentiate signalling. Thus, it seems that Mal phosphorylation by Btk allows a transient activation of downstream signalling pathways before Mal is targeted for degradation which would, in effect, aid in control of the response.88

It was also recently found that Mal is cleaved by caspase-1. A mutant form of Mal that could not undergo caspase-1-mediated cleavage was unable to signal and acted as a dominant negative inhibitor of TLR2 and TLR4 signalling, suggesting that Mal cleavage may be necessary for its ability to signal. Cleavage of Mal by caspase-1 results in a 4 kDa portion of Mal being removed from the TIR domain.89 It could be postulated that this would cause a conformational change of the TIR domain or perhaps expose residues necessary for the interaction of Mal with TLR2, TLR4 or MyD88.

In addition, Mal was shown to interact via its TIR domain with protein kinase C-delta (PKCdelta).90 Depletion of PKCdelta from Raw264.7 cells abolished phosphorylation of p38 MAPK, IKK and IkappaB downstream of TLR4,90 suggesting that the interaction of PKCdelta with the TIR domain of Mal promotes the ability of Mal to activate signalling.

Finally, a variant of Mal was discovered in which the serine at position 180 is mutated to a leucine. Interestingly, heterozygous carriage of this variant was found to confer a protective effect against several infectious diseases, and it was shown that the S180L mutation prevented signalling by TLR2. It was predicted that Ser180 is located on the surface of the TIR domain, adjacent to the DD loop, a motif that has been implicated in the interaction of Mal with TLR2. The presence of a leucine at position 180 prevented the association of Mal with TLR2 which explains the inability of this mutant to signal. It is suggested that heterozygosity at S180L confers a protective phenotype by balancing the inflammatory response.91

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TRIF

TRIF has also been shown to be subject to negative regulation. TRAF1 was recently shown to be a negative regulator of TRIF. The TIR domain of TRIF was involved in the interaction with the TRAF-C domain of TRAF1, and inhibition most likely occurs through sequestration of the TIR domain. Given the importance of TLR signalling in the initial response to viruses, it is not surprising that viruses have evolved mechanisms to disrupt TLR signalling. Indeed, vaccinia virus (VV) encodes a TIR domain containing protein, A46R, which sequesters TRIF via homophilic TIR domain interactions and inhibited TRIF-induced IRF3 activation. A46R also targets MyD88 and TRAM, which like TRIF, also play a role in inducing antiviral signalling pathways. In addition, a serine protease of hepatitis C virus (HCV) is known to cause proteolysis of TRIF. As already discussed, SARM interacts with TRIF and specifically inhibits TRIF-signalling pathways.

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TRAM

In an analogous situation to Mal, TRAM is targeted to the plasma membrane, although the mechanism by which this occurs is different. TRAM contains a putative N-terminal myristoylation site (Figure 2), which when mutated, causes the dissociation of TRAM from the plasma membrane indicating that TRAM myristoylation serves to target it to the membrane. This mutant TRAM also failed to support IRF3 or NF-kappaB signalling, which suggests that the membrane localization of TRAM is essential for signalling to occur. Membrane localization of TRAM is probably required in order for it to recruit TRIF to the membrane as the only known function attributed to TRAM is that of a bridging adaptor between TLR4 and TRIF.92

TRAM is also subject to regulation by phosphorylation. It was found that TRAM was phosphorylated on serine-16 by PKCalt epsilon upon LPS stimulation (Figure 2). This phosphorylation event is necessary for TRAM signalling as TRAM S16A abolished the ability of the adaptor to activate IRF3 or NF-kappaB. TRAM phosphorylation by PKCalt epsilon also correlates with its depletion from the membrane although the significance of this event is unknown. One possibility is that phosphorylation of TRAM by PKCalt epsilon causes it to become activated at the membrane before dissociation, perhaps due to a conformational change in the protein.93 However, unlike the case where Mal is phosphorylated by Btk, the phosphorylation site on TRAM is not in the TIR domain. In fact Serine-16 is quite removed from the TIR domain in the primary sequence, making it more unlikely that phosphorylation at this residue should cause a conformational change of the TIR domain. Another scenario is that phosphorylation at this membrane proximal site causes dissociation of TRAM from the membrane, thereby allowing TRAM to interact with downstream signalling molecules.93

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Conclusion and future perspectives

It has been known for some time that differential recruitment of adaptors to the intracellular domains of TLRs allows the activation of distinct pathways. However, recent studies have indicated that this differential recruitment is largely due to inherent differences of the adaptor and TLR TIR domains. Furthermore, adaptors undergo various types of modification which may serve to potentiate or abrogate signalling, possibly by altering the TIR domain conformation. Indeed, both Mal and TRAM are subject to regulation by covalent modification. Mal, in particular, is very highly regulated which allows for a robust response to TLR2 and TLR4 ligands which is quickly controlled to avoid immunopathology. The TIR domain containing adaptors, therefore, provide a high degree of specificity to TLR-signalling pathways, mainly due to the structure of their TIR domains and the modifications they undergo. Future structural studies will no doubt identify the exact interfaces involved in the receptor adaptor interface, and this knowledge will aid in the design of pharmacologic agents to alter TIR domain interactions.

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Acknowledgements

We thank Science Foundation Ireland, Health Research Board of Ireland and Irish Research Council for Science, Engineering and Technology for providing financial support.

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