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

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.     

Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88

The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.     

The Brucella TIR-like protein TcpB interacts with the death domain of MyD88

The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB-MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88(DD)-MyD88(DD), MyD88(DD)-MyD88(TIR) and MyD88(DD)-MyD88 interactions but not MyD88-MyD88 or MyD88(TIR)-MyD88(TIR) interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD-TIR domain interface.     

A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins

The Toll-like receptor 4 (TLR4) is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR) signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3) and nuclear factor kappaB (NFkappaB) respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.     

Inhibition of Toll-like receptors TLR4 and 7 signaling pathways by SIGIRR: A computational approach

Toll-like receptors (TLRs) belong to the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily which is defined by a common cytoplasmic Toll/interleukin-1 receptor (TIR) domain. TLRs recognize pathogen-associated molecular patterns and initiate an intracellular kinase cascade to trigger an immediate defensive response. SIGIRR (single immunoglobulin interleukin-1 receptor-related molecule), another member of the TLR/IL-1R superfamily, acts as a negative regulator of MyD88-dependent TLR signaling. It attenuates the recruitment of MyD88 adaptors to the receptors with its intracellular TIR domain. Thus, SIGIRR is a highly important molecule for the therapy of autoimmune diseases caused by TLRs. So far, the structural mechanism of interactions between SIGIRR, TLRs and adaptor molecules is unclear. To develop a working hypothesis for this interaction, we constructed three-dimensional models for the TIR domains of TLR4, TLR7, MyD88 and SIGIRR based on computational modeling. Through protein–protein docking analysis, we developed models of essential complexes involved in the TLR4 and 7 signaling and the SIGIRR inhibiting processes. We suggest that SIGIRR may exert its inhibitory effect through blocking the molecular interface of TLR4, TLR7 and the MyD88 adaptor mainly via its BB-loop region.     

Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins

Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.     

TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88

MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.     

A novel TLR2‐triggered signalling crosstalk synergistically intensifies TNF‐mediated IL‐6 induction

Toll‐like receptors (TLR) recognize pathogens and trigger the production of vigorous pro‐inflammatory cytokines [such as tumour necrosis factor (TNF)] that induce systemic damages associated with sepsis and chronic inflammation. Cooperation between signals of TLR and TNF receptor has been demonstrated through the participation of TNF receptor 1 (TNFR) adaptors in endotoxin tolerance. Here, we identify a TLR2‐mediated synergy, through a MyD88‐independent crosstalk, which enhances subsequent TNF‐mediated nuclear factor‐kappa B activation and interleukin‐6 induction. Membrane‐associated adaptor MAL conduces the link between TNF receptor‐associated factor 6 (TRAF6) and TNFR‐associated death domain, leading to a distinctive K63‐ubiquitinylated TRAF6 recruitment into TNFR complex. In summary, our results reveal a novel route of TLR signal that synergistically amplifies TNF‐mediated responses, indicating an innovative target for inflammation manipulation.     

Toll/Interleukin-1 Receptor Domain Dimers as the Platform for Activation and Enhanced Inhibition of Toll-like Receptor Signaling

TIR (Toll/IL-1 receptor) domains mediate interactions between TLR (Toll-like) or IL-1 family receptors and signaling adapters. While homotypic TIR domain interactions mediate receptor activation they are also usurped by microbial TIR domain containing proteins for immunosuppression. Here we show the role of a dimerized TIR domain platform for the suppression as well as for the activation of MyD88 signaling pathway. Coiled-coil dimerization domain, present in many bacterial TCPs, potently augments suppression of TLR/IL-1R signaling. The addition of a strong coiled-coil dimerization domain conferred the superior inhibition against the wide spectrum of TLRs and prevented the constitutive activation by a dimeric TIR platform. We propose a molecular model of MyD88-mediated signaling based on the dimerization of TIR domains as the limiting step.     

Toll receptors and pathogen resistance

Toll receptors in insects, mammals and plants are key players that sense the invasion of pathogens. Toll-like receptors (TLRs) in mammals have been established to detect specific components of bacterial and fungal pathogens. Furthermore, recent evidence indicates that TLRs are involved in the recognition of viral invasion. Signalling pathways via TLRs originate from the conserved Toll/IL-1 receptor (TIR) domain. The TIR domain-containing MyD88 acts as a common adaptor that induces inflammatory cytokines; however, there exists a MyD88-independent pathway that induces type I IFNs in TLR4 and TLR3 signalling. Another TIR domain-containing adaptor, TIRAP/Mal has recently been shown to mediate the MyD88-dependent activation in the TLR4 and TLR2 signalling pathway. Thus, individual TLRs may have their own signalling systems that characterize their specific activities.     

Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.     

Toll IL-1 receptors differ in their ability to promote the stabilization of adenosine and uridine-rich elements containing mRNA

Several ligands for Toll IL-1R (TIR) family are known to promote stabilization of a subset of short-lived mRNAs containing AU-rich elements (AREs) in their 3' untranslated regions. It is now evident however, that members of the TIR family may use distinct intracellular signaling pathways to achieve a spectrum of biological end points. Using human embryonic kidney 293 cells transfected to express different TIRs we now report that signals initiated through IL-1R1 or TLR4 but not TLR3 can promote the stabilization of unstable chemokine mRNAs. Similar results were obtained when signaling from endogenous receptors was examined using a mouse endothelial cell line (H5V). The ability of TIR family members to stabilize ARE-containing mRNAs results from their differential use of signaling adaptors MyD88, MyD88 adaptor-like protein, Toll receptor IFN-inducing factor (Trif), and Trif-related adaptor molecule. Overexpression of MyD88 or MyD88 adaptor-like protein was able to promote enhanced stability of ARE-containing mRNA, whereas Trif and Trif-related adaptor molecule exhibited markedly reduced capacity. Hence the ability of TIRs to signal stabilization of mRNA appears to be linked to the MyD88-dependent signaling pathway.     

Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family

The Toll/interleukin-1 (IL-1) receptor (TIR) family comprises two groups of transmembrane proteins, which share functional and structural properties. The members of the IL-1 receptor (IL-1R) subfamily are characterized by three extracellular immunoglobulin (Ig)-like domains. They form heterodimeric signaling receptor complexes consisting of receptor and accessory proteins. The members of the Toll-like receptor (TLR) subfamily recognize alarm signals that can be derived either from pathogens or the host itself. TLRs possess leucine-rich repeats in their extracellular part. TLRs can form dimeric receptor complexes consisting of two different TLRs or homodimers in the case of TLR4. The TLR4 receptor complex requires supportive molecules for optimal response to its ligand lipopolysaccharide (LPS). A hallmark of the TIR family is the cytoplasmic TIR domain that is indispensable for signal transduction. The TIR domain serves as a scaffold for a series of protein-protein interactions which result in the activation of a unique signaling module consisting of MyD88, interleukin-1 receptor associated kinase (IRAK) family members and Tollip, which is used exclusively by TIR family members. Subsequently, several central signaling pathways are activated in parallel, the activation of NFkappaB being the most prominent event of the inflammatory response. Recent developments indicate that in addition to the common signaling module MyD88/IRAK/Tollip, other molecules can modulate signaling by TLRs, especially of TLR4, resulting in differential biological answers to distinct pathogenic structures. Subtle differences in TLR signaling pathways are now becoming apparent, which reveal how the innate immune system decides at a very early stage the direction in which the adaptive immune response will develop. The creation of pathogen-specific mediator environments by dendritic cells defines whether a cellular or humoral response will be activated in response to the pathogen.     

1H, 13C, and 15N resonance assignment of the TIR domain of human MyD88

Myeloid differentiating factor 88 (MyD88) is one of a critical adaptor molecule in the Toll-like receptor (TLR) signaling pathway. The TIR domain of MyD88 serves as a protein–protein interaction module and interacts with other TIR-containing proteins such as Mal (MyD88 adaptor-like) and Toll-like receptor 4 to form signal initiation complexes. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the TIR domain of MyD88. The resonance assignments obtained in this work will contribute to the study of heteromeric TIR–TIR interactions between MyD88 and TIR-containing receptors or adaptors.     

Investigating the effect of key mutations on the conformational dynamics of toll‐like receptor dimers through molecular dynamics simulations and protein structure networks

The Toll‐like receptors (TLRs) are critical components of the innate immune system due to their ability to detect conserved pathogen‐associated molecular patterns, present in bacteria, viruses, and other microorganisms. Ligand detection by TLRs leads to a signaling cascade, mediated by interactions among TIR domains present in the receptors, the bridging adaptors and sorting adaptors. The BB loop is a highly conserved region present in the TIR domain and is crucial for mediating interactions among TIR domain‐containing proteins. Mutations in the BB loop of the Toll‐like receptors, such as the A795P mutation in TLR3 and the P712H mutation (Lps^d mutation) in TLR4, have been reported to disrupt or alter downstream signaling. While the phenotypic effect of these mutations is known, the underlying effect of these mutations on the structure, dynamics and interactions with other TIR domain‐containing proteins is not well understood. Here, we have attempted to investigate the effect of the BB loop mutations on the dimer form of TLRs, using TLR2 and TLR3 as case studies. Our results based on molecular dynamics simulations, protein–protein interaction analyses and protein structure network analyses highlight significant differences between the dimer interfaces of the wild‐type and mutant forms and provide a logical reasoning for the effect of these mutations on adaptor binding to TLRs. Furthermore, it also leads us to propose a hypothesis for the differential requirement of signaling and bridging adaptors by TLRs. This could aid in further understanding of the mechanisms governing such signaling pathways.     

A putative Toll/interleukin‐1 receptor domain protein from Helicobacter pylori is dimeric in solution and interacts with human Toll‐like receptor adaptor myeloid differentiation primary response 88

Helicobacter pylori, an important human pathogen, is capable of causing persistent infection with minimal immune response. The first line of defense during H. pylori infection is through gastric epithelial cells that present TLR, A family of bacterial proteins that share homology with the Toll/IL‐1 receptor (TIR) domain were identified. Bacterial TIR proteins (BTP) mimic human TIR domain proteins and act on myeloid differentiation primary response gene 88 (MyD88) signaling pathways to suppress TLR signaling. H. pylori may also produce a similar protein. A putative H. pylori BTP was found based on sequence homology. The corresponding gene hp1437 was inserted into an expression vector in fusion with an N‐terminal cleavable 6his‐tag. The recombinant protein, 6his‐HP1437, was purified using nickel affinity chromatography with a yield of 8 mg/L culture. Oligomerization of HP1437 was investigated by size‐exclusion chromatography. It was found that HP1437 forms dimers in solution similar to other BTPs. Furthermore, glutathione S‐transferase pull down assays identified an interaction between HP1437 and human TIR domain adaptor MyD88. These findings suggest that HP1437 has the characteristic features of BTPs and may play a direct role in reducing immune response against H. pylori by binding to MyD88 and pave the way for an in‐depth characterization of this putative novel H. pylori virulence factor.     

Structural insights into TIR domain specificity of the bridging adaptor Mal in TLR4 signaling

MyD88 adaptor-like protein (Mal) is a crucial adaptor that acts as a bridge to recruit the MyD88 molecule to activated TLR4 receptors in response to invading pathogens. The specific assembly of the Toll/interleukin-1 receptor (TIR) domains of TLR4, Mal and MyD88 is responsible for proper signal transduction in the TLR4 signaling pathway. However, the molecular mechanism for the specificity of these TIR domains remains unclear. Here, we present the crystal structure of the TIR domain of the human Mal molecule (Mal-TIR) at a resolution of 2.4 Å. Unexpectedly, Mal-TIR exhibits an extraordinarily long AB loop, but no αB helix or BB loop, distinguishing it from other TIR domains. More importantly, the Mal-TIR AB loop is capable of mediating direct binding to the TIR domains of TLR4 and MyD88 simultaneously. We also found that Mal-TIR can form a back-to-back dimer that may resemble the dimeric assembly of the entire Mal molecule. Our data demonstrate the bridge role of the Mal-TIR domain and provide important information about TIR domain specificity.