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.     

Plant immune signaling: Advancing on two frontiers

Plants have evolved multiple defense strategies to cope with pathogens, among which plant immune signaling that relies on cell-surface localized and intracellular receptors takes fundamental roles. Exciting breakthroughs were made recently on the signaling mechanisms of pattern recognition receptors(PRRs) and intracellular nucleotide-binding site(NBS) and leucine-rich repeat(LRR)domain receptors(NLRs). This review summarizes the current view of PRRs activation, emphasizing the most recent discoveries about PRRs’ dynamic regulation and signaling mechanisms directly leading to downstream molecular events including mitogen-activated protein kinase(MAPK) activation and calcium(Ca2+) burst. Plants also have evolved intracellular NLRs to perceive the presence of specific pathogen effectors and trigger more robust immune responses. We also discuss the current understanding of the mechanisms of NLR activation, which has been greatly advanced by recent breakthroughs including structures of the first full-length plant NLR complex, findings of NLR sensor-helper pairs and novel biochemical activity of Toll/interleukin-1 receptor(TIR) domain.     

The crystal structure of a TIR domain from Arabidopsis thaliana reveals a conserved helical region unique to plants

Plants use a highly evolved immune system to exhibit defense response against microbial infections. The plant TIR domain, together with the nucleotide‐binding (NB) domain and/or a LRR region, forms a type of molecule, named resistance (R) proteins, that interact with microbial effector proteins and elicit hypersensitive responses against infection. Here, we report the first crystal structure of a plant TIR domain from Arabidopsis thaliana (AtTIR) solved at a resolution of 2.0 Å. The structure consists of five β‐strands forming a parallel β‐sheet at the core of the protein. The β‐strands are connected by a series of α‐helices and the overall fold mimics closely that of other mammalian and bacterial TIR domains. However, the region of the αD‐helix reveals significant differences when compared with other TIR structures, especially the αD3‐helix that corresponds to an insertion only present in plant TIR domains. Available mutagenesis data suggest that several conserved and exposed residues in this region are involved in the plant TIR signaling function.     

A bioinformatic approach to the identification of bacterial proteins interacting with Toll-interleukin 1 receptor-resistance (TIR) homology domains

Members of the Toll-like receptor (TLR) family are currently under intense scrutiny for their role in the sampling and recognition of pathogens. It has already been reported that both vaccinia virus and Yersinia spp. express proteins that help them evade the TLR mediated immune response, acting through the Toll-interleukin-1 receptor-resistance (TIR) domain and leucine-rich repeat region of the host TLRs respectively. The TIR domain is involved in the dimerisation of the TLRs and their complexation with their adapter molecules. We tested here the hypothesis that bacteria have the ability to secrete proteins containing similar motifs to the intracellular TIR domains that are involved in the TIR-TIR interaction necessary for the subsequent signal transmission. Based upon their sequence homology, proteins expressing TIRs have been divided into three sub-classes, based around the TLRs, the TLR adapter proteins, and the interleukin-1 and -18 adapter proteins. The highly conserved regions from these separate sub-families were then used to identify similar bacterial proteins. The bacterial proteins identified were then included in an iterative MEME-BLAST process to broaden the search. Tollip, a known TLR antagonist and adapter protein, was included in this investigation although it does not fit into any of the three sub-classes outlined above. If suitable bacterial proteins had been identified, it would signify that certain bacteria had evolved a mechanism to aid them in avoiding detection by the innate immune system acting through the TIR domains. At this stage one has to conclude that there is no evidence currently available suggesting such a mechanism, when using the strategy applied here.     

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu¹⁸³, Ser²⁴⁴, and Arg²⁸⁸ impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88_151–189 and GFP-MyD88_168–189), comprising the Glu¹⁸³ residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.     

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.     

S-nitrosylation of surfactant protein D as a modulator of pulmonary inflammation

Background

Surfactant protein D (SP-D) is a member of the family of proteins termed collagen-like lectins or “collectins” that play a role in non-antibody-mediated innate immune responses [1]. The primary function of SP-D is the modulation of host defense and inflammation [2].

Scope of review

This review will discuss recent findings on the physiological importance of SP-D S-nitrosylation in biological systems and potential mechanisms that govern SP-D mediated signaling.

Major conclusions

SP-D appears to have both pro- and anti-inflammatory signaling functions.SP-D multimerization is a critical feature of its function and plays an important role in efficient innate host defense. Under baseline conditions, SP-D forms a multimer in which the N-termini are hidden in the center and the C-termini are on the surface. This multimeric form of SP-D is limited in its ability to activate inflammation. However, NO can modify key cysteine residues in the hydrophobic tail domain of SP-D resulting in a dissociation of SP-D multimers into trimers, exposing the S-nitrosylated N-termini. The exposed S-nitrosylated tail domain binds to the calreticulin/CD91 receptor complex and initiates a pro-inflammatory response through phosphorylation of p38 and NF-κB activation [3,4]. In addition, the disassembled SP-D loses its ability to block TLR4, which also results in activation of NF-κB.

General significance

Recent studies have highlighted the capability of NO to modify SP-D through S-nitrosylation, causing the activation of a pro-inflammatory role for SP-D [3]. This represents a novel mechanism both for the regulation of SP-D function and NO's role in innate immunity, but also demonstrates that the S-nitrosylation can control protein function by regulating quaternary structure. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation.     

Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation

The Toll/interleukin-1 receptor (TIR) domain occurs in animal and plant immune receptors. In the animal Toll-like receptors, homodimerization of the intracellular TIR domain is required for initiation of signaling cascades leading to innate immunity. By contrast, the role of the TIR domain in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant immune resistance proteins is poorly understood. L6 is a TIR-NB-LRR resistance protein from flax (Linum usitatissimum) that confers resistance to the flax rust phytopathogenic fungus (Melampsora lini). We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming?a homodimer. Analysis of the structure combined with site-directed mutagenesis suggests that self-association is a requirement for immune signaling and reveals distinct surface regions involved in self-association, signaling, and autoregulation.     

The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3C^pro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3C^pro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3C^pro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3C^pro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.     

Phytophthora infestans RXLR Effector PexRD2 Interacts with Host MAPKKKε to Suppress Plant Immune Signaling

Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity–related signaling.     

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.     

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.     

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.     

Divergent signaling requirements of dSARM in injury-induced degeneration and developmental glial phagocytosis

Elucidating signal transduction mechanisms of innate immune pathways is essential to defining how they elicit distinct cellular responses. Toll-like receptors (TLR) signal through their cytoplasmic TIR domains which bind other TIR domain-containing adaptors. dSARM/SARM1 is one such TIR domain adaptor best known for its role as the central axon degeneration trigger after injury. In degeneration, SARM1’s domains have been assigned unique functions: the ARM domain is auto-inhibitory, SAM-SAM domain interactions mediate multimerization, and the TIR domain has intrinsic NAD⁺ hydrolase activity that precipitates axonal demise. Whether and how these distinct functions contribute to TLR signaling is unknown. Here we show divergent signaling requirements for dSARM in injury-induced axon degeneration and TLR-mediated developmental glial phagocytosis through analysis of new knock-in domain and point mutations. We demonstrate intragenic complementation between reciprocal pairs of domain mutants during development, providing evidence for separability of dSARM functional domains in TLR signaling. Surprisingly, dSARM’s NAD⁺ hydrolase activity is strictly required for both degenerative and developmental signaling, demonstrating that TLR signal transduction requires dSARM’s enzymatic activity. In contrast, while SAM domain-mediated dSARM multimerization is important for axon degeneration, it is dispensable for TLR signaling. Finally, dSARM functions in a linear genetic pathway with the MAP3K Ask1 during development but not in degenerating axons. Thus, we propose that dSARM exists in distinct signaling states in developmental and pathological contexts.     

The role of NDR1 in pathogen perception and plant defense signaling

The biochemical and cellular function of NDR1 in plant immunity and defense signaling has long remained elusive. Herein, we describe a novel role for NDR1 in both pathogen perception and plant defense signaling, elucidated by exploring a broader, physiological role for NDR1 in general stress responses and cell wall adhesion. Based on our predictive homology modeling, coupled with a structure-function approach, we found that NDR1 shares a striking similarity to mammalian integrins, well-characterized for their role in mediating the interaction between the extracellular matrix and stress signaling. ndr1-1 mutant plants exhibit higher electrolyte leakage following pathogen infection, compared to wild type Col-0. In addition, we observed an altered plasmolysis phenotype, supporting a role for NDR1 in maintaining cell wall-plasma membrane adhesions through mediating fluid loss under stress.Key words: NDR1, integrin, RGD, plant defenseNON-RACE SPECIFIC DISEASE RESISTANCE-1(NDR1) was first identified as playing an essential role in plant defense activation following the perception of the bacterial phytopathogen Pseudomonas syringae pv tomato DC3000.¹ Over the past decade, the role of NDR1 in plant defense signaling has emerged through the elucidation of the genetic interactions in which NDR1 participates. This includes the activation of the coiled-coil nucleotide binding site leucine rich repeat (CC-NB-LRR) family of resistance (R) proteins. In parallel, the function of ENHANCED DISEASE SUCEPTIBILITY-1 (EDS1 ²) has been extensively described through its genetic and biochemical relationship with the activation of Toll-Interleukin Receptor (TIR)-NB-LRR R-proteins.³ As central activators required for defense signaling, NDR1 and EDS1 represent critical nodes required for the activation of host resistance.     

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.