Crystal Structures of the Toll/Interleukin-1 Receptor (TIR) Domains from the Brucella Protein TcpB and Host Adaptor TIRAP Reveal Mechanisms of Molecular Mimicry

The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys⁸⁹ and Cys¹³⁴. A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.     

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.     

Scaffolding role of TcpB in disrupting TLR4-Mal interactions: Three to tango

Toll/interleukin-1 like receptors (TLRs) are membrane-spanning proteins crucially involved in innate immunity. On activation, the cytoplasmic toll/interleukin-1 receptor (TIR) domains of these receptors undergo homo- or heterodimerization. Brucella sp. are bacterial pathogens that affect the immune system by suppressing the TLR signaling pathway. They enact this by encoding a TIR domain–containing protein, TcpB, which suppresses NF-κB activation and proinflammatory cytokine secretion mediated by TLR4 receptors. TcpB has been shown to target the Mal-mediated pathway to suppress TLR signaling. The recent identification of its mechanism of interference with TLR4 signaling involving Mal prompted us to further study the structural aspects of TcpB binding with TLR4 and Mal. Our triprotein model displays the overall scaffolding role of TcpB in anchoring TLR4 and Mal thereby inhibiting their interaction leading to the attenuation of the TLR4 pathway.     

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.     

Structure of the Toll/interleukin 1 receptor (TIR) domain of the immunosuppressive Brucella effector BtpA/Btp1/TcpB

BtpA/Btp1/TcpB is a virulence factor produced by Brucella species that possesses a Toll interleukin-1 receptor (TIR) domain. Once delivered into the host cell, BtpA interacts with MyD88 to interfere with TLR signalling and modulates microtubule dynamics. Here the crystal structure of the BtpA TIR domain at 3.15 Å is presented. The structure shows a dimeric arrangement of a canonical TIR domain, similar to the Paracoccus denitrificans Tir protein but secured by a unique long N-terminal α-tail that packs against the TIR:TIR dimer. Structure-based mutations and multi-angle light scattering experiments characterized the BtpA dimer conformation in solution. The structure of BtpA will help with studies to understand the mechanisms involved in its interactions with MyD88 and with microtubules.     

Biochemical and functional analysis of TIR domain containing protein from Brucella melitensis

Toll/interleukin-1 like receptors are evolutionarily conserved proteins in eukaryotes that play crucial role in pathogen recognition and innate immune responses. Brucella are facultative intracellular bacterial pathogens causing brucellosis in animal and human hosts. Brucella behave as a stealthy pathogen by evading the immune recognition or suppressing the TLR signaling cascades. Brucella encode a TIR domain containing protein, TcpB, which suppresses NF-κB activation as well as pro-inflammatory cytokine secretion mediated by TLR2 and TLR4 receptors. TcpB targets the TIRAP mediated pathway to suppress TLR signaling. With the objective of detailed characterization, we have over expressed and purified TcpB from Brucella melitensis in native condition. The purified protein exhibited lipid-binding properties and cell permeability. NF-κB inhibition property of endogenous TcpB has also been demonstrated. The data provide insight into the mechanism of action of TcpB in the intracellular niche of Brucella.     

A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain‐containing protein (PumA) of the multi‐drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF‐κB, a property transferable to non‐PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll‐like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin‐associated protein 1 (UBAP1), a component of the endosomal‐sorting complex required for transport I (ESCRT‐I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.     

The interleukin 1 (IL-1) receptor accessory protein Toll/IL-1 receptor domain: analysis of putative interaction sites in vitro mutagenesis and molecular modeling

The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal alpha-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527-534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants delta528-534 and delta527-533, whereas the C-terminal beta-strand and/or alpha-helix is displaced in the nonresponsive mutant delta527-534.     

The crystal structure of the human toll-like receptor 10 cytoplasmic domain reveals a putative signaling dimer

The Toll/interleukin-1 receptor (TIR) domain is a highly conserved signaling domain found in the intracellular regions of Toll-like receptors (TLRs), in interleukin-1 receptors, and in several cytoplasmic adaptor proteins. TIR domains mediate receptor signal transduction through recruitment of adaptor proteins and play critical roles in the innate immune response and inflammation. This work presents the 2.2A crystal structure of the TIR domain of human TLR10, revealing a symmetric dimer in the asymmetric unit. The dimer interaction surface contains residues from the BB-loop, DD-loop, and alphaC-helix, which have previously been identified as important structural motifs for signaling in homologous TLR receptors. The interaction surface is extensive, containing a central hydrophobic patch surrounded by polar residues. The BB-loop forms a tight interaction, where a range of consecutive residues binds in a pocket formed by the reciprocal BB-loop and alphaC-helix. This pocket appears to be well suited for binding peptide substrates, which is consistent with the notion that peptides and peptide mimetics of the BB-loop are inhibitors for TLR signaling. The TLR10 structure is in good agreement with available biochemical data on TLR receptors and is likely to provide a good model for the physiological dimer.     

LRRC19, a novel member of the leucine-rich repeat protein family, activates NF-κB and induces expression of proinflammatory cytokines

We have identified a new functional transmembrane receptor, LRRC19 (leucine-rich repeat containing 19), that belongs to the LRR protein family. LRRC19’s central core has four analogous LRR repeating modules in a juxtaposed array and a casein kinase (CK2) phosphorylation site in the cytoplasmic domain. LRRC19 mRNA was found in the kidney, spleen and intestine of adult mice using both RT-PCR and in situ hybridization. LRRC19 does not contain a cytoplasmic Toll/IL-1 receptor (TIR) domain but was able to activate NF-κB and induce production of proinflammatory cytokines. LRRC19 shares a close evolutionary relationship with multiple Toll-like receptors (TLRs), especially TLR3. Importantly, the TLR3 ligand, as well as other TLR ligands, significantly promoted the expression of proinflammatory cytokines and the activation of NF-κB by LRRC19. Thus, LRRC19 may play an important role in inducing innate immune responses in certain tissues such as the kidney.     

Probing subtle conformational changes induced by phosphorylation and point mutations in the TIR domains of TLR2 and TLR3

Extensive research performed on Toll‐like receptor (TLR) signaling has identified residues in the Toll/interleukin‐1 receptor (TIR) domains that are essential for its proper functioning. Among these residues, those in BB loop are particularly significant as single amino acid mutations in this region can cause drastic changes in downstream signaling. However, while the effect of these mutations on the function is well studied (like the P681H mutation in TLR2, the A795P mutation in TLR3, and the P714H mutation in TLR4), their influence on the dynamics and inter‐residue networks is not well understood. The effects of local perturbations induced by these mutations could propagate throughout the TIR domain, influencing interactions with other TIR domain‐containing proteins. The identification of these subtle changes in inter‐residue interactions can provide new insights and structural rationale for how single‐point mutations cause drastic changes in TIR–TIR interactions. We employed molecular dynamics simulations and protein structure network (PSN) analyses to investigate the structural transitions with special emphasis on TLR2 and TLR3. Our results reveal that phosphorylation of the Tyr 759 residue in the TIR domain of TLR3 introduces rigidity to its BB loop. Subtle differences in the intra BB loop hydrogen bonding network between TLR3 and TLR2 are also observed. The PSN analyses indicate that the TIR domain is highly connected and pinpoints key differences in the inter‐residue interactions between the wild‐type and mutant TIR domains, suggesting that TIR domain structure is prone to allosteric effects, consistent with the current view of the influence of allostery on TLR signaling.     

Crystal Structure of TIR Domain of TLR6 Reveals Novel Dimeric Interface of TIR–TIR Interaction for Toll-Like Receptor Signaling Pathway

Toll-like receptors (TLRs) are responsible for recognition of particular pathogens during the innate immune response and cytoplasmic Toll/interleukin-1 receptor (TIR) domain responsible for downstream signaling. TLR6 working with TLR2 can detect bacterial lipoprotein leading signal for nuclear factor-kappaB activation for immune response. To better understand TLR-mediated signaling event in the innate immune system, in this study, we report the first crystal structure of the TIR domain of TLR6 at 2.2 Å resolution. Our structure reveals novel homo-dimerization interfaces, which might be a critical for the interaction with TIR-containing adaptor proteins and itself. We also report structural similarities and differences of TLR6 with those of other TIR domains, which may be functionally relevant.     

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.     

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.     

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.     

dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8

Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2-TNF receptor-associated factor (TRAF)2-TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.     

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.     

Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL

The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-A resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix alphaD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor.