Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Toll/interleukin-1 receptor domains in bacterial and plant immunity.

The Toll/interleukin-1 receptor (TIR) domain is found in animal, plant, and bacterial immune systems. It was first described as a protein-protein interaction module mediating signalling downstream of the Toll-like receptor and interleukin-1 receptor families in animals. However, studies of the pro-neurodegenerative protein sterile alpha and TIR motif containing 1, plant immune receptors, and many bacterial TIR domain-containing proteins revealed that TIR domains have enzymatic activities and can produce diverse nucleotide products using nicotinamide adenine dinucleotide (NAD+) or nucleic acids as substrates. Recent work has led to key advances in understanding how TIR domain enzymes work in bacterial and plant immune systems as well as the function of their signalling molecules.

Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling.

Cyclic adenosine diphosphate (ADP)-ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD+) hydrolysis. We show that v-cADPR (2'cADPR) and v2-cADPR (3'cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2'cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3'cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3'cADPR in bacteria as an antiviral and plant immunity-suppressing signaling molecule.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence.

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.

Cyclic nucleotide-induced helical structure activates a TIR immune effector.

Cyclic nucleotide signalling is a key component of antiviral defence in all domains of life. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway1, which originated in bacteria2. These defence systems require a sensor domain to bind the cyclic nucleotide and are often coupled with an effector domain that, when activated, causes cell death by destroying essential biomolecules3. One example is the Toll/interleukin-1 receptor (TIR) domain, which degrades the essential cofactor NAD+ when activated in response to infection in plants and bacteria2,4,5 or during programmed nerve cell death6. Here we show that a bacterial antiviral defence system generates a cyclic tri-adenylate that binds to a TIR-SAVED effector, acting as the 'glue' to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organize and complete a composite active site, allowing NAD+ degradation. Activation requires extended filament formation, both in vitro and in vivo. Our study highlights an example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

Cryo-EM structure of an active bacterial TIR-STING filament complex.

Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1-4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4-13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)-STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR-STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR-STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.

The Nucleotide Revolution: Immunity at the Intersection of Toll/Interleukin-1 Receptor Domains, Nucleotides, and Ca2.

The discovery of the enzymatic activity of the toll/interleukin-1 receptor (TIR) domain protein SARM1 five years ago preceded a flood of discoveries regarding the nucleotide substrates and products of TIR domains in plants, animals, bacteria, and archaea. These discoveries into the activity of TIR domains coincide with major advances in understanding the structure and mechanisms of NOD-like receptors and the mutual dependence of pattern recognition receptor- and effector-triggered immunity (PTI and ETI, respectively) in plants. It is quickly becoming clear that TIR domains and TIR-produced nucleotides are ancestral signaling molecules that modulate immunity and that their activity is closely associated with Ca2+ signaling. TIR domain research now bridges the separate disciplines of molecular plant- and animal-microbe interactions, neurology, and prokaryotic immunity. A cohesive framework for understanding the role of enzymatic TIR domains in diverse organisms will help unite the research of these disparate fields. Here, we review known products of TIR domains in plants, animals, bacteria, and archaea and use context gained from animal and prokaryotic TIR domain systems to present a model for TIR domains, nucleotides, and Ca2+ at the intersection of PTI and ETI in plant immunity. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Interleukin-1α, interleukin-1ß and interleukin-1 receptor antagonist share a common U-shaped recognition epitope on interleukin-1 receptor surface.

Interleukin-1 (IL-1) plays a central role in the regulation of immune and inflammatory responses. There are two forms of IL-1 agonists (IL-1α and IL-1ß) and one form of IL-1 antagonist (IL-1Ra); they share a similar binding mode to the IL-1 receptor (IL-1R) but exhibit opposite biological functions on the receptor. In this study, the intermolecular interactions of IL-1R receptors with IL-1α, IL-1ß and IL-1Ra ligands were systematically investigated at structural, energetic and dynamic levels. It was found that the receptor primarily adopts a U-shaped, double-stranded and linear/conformational-hybrid epitope to commonly interact with the three ligands. The epitope covers a common protein segment (residues 107-127), which is fully located within the C2T2 subdomain of the IL-1R extracellular domain and contributes ~40% to the total binding energy of IL-1R/ligand association. The epitope is natively folded into an ordered conformation in the IL-1R protein context but would become largely disordered out of the context. Here, we adopted a disulfide bridge to staple U-shaped epitope-derived peptides, which can be effectively constrained into a native-like conformation and thus exhibit an improved affinity to ligands as compared to their unstapled counterpart, with affinity increase by up to ~15-fold. These disulfide bridges were designed to point out of ligand/peptide complex interface and thus would not disrupt the direct complex interaction. Energetic decomposition imparted that the stapling has only a modest influence on the interaction enthalpy and desolvation effect of ligand/peptide binding, but can substantially reduce entropy penalty upon the binding. For a peptide, the stapling-addressed entropic reduction can be roughly regarded as a constant, which only improves peptide affinity to these ligands, but does not change peptide selectivity over different ligands.

Interleukin-1 Receptor-Like 2: One Receptor, Three Agonists, and Many Implications.

The interleukin (IL)-1 superfamily of cytokines comprises 11 pro- and anti-inflammatory cytokines, which play essential roles during the immune response. Several pathogenic pathways are initiated by IL-1RL2 (interleukin 1 receptor-like 2) signaling, also known as IL-36R, in the skin, lungs, and gut. IL-36 cytokines promote the secretion of proinflammatory cytokines and chemokines, upregulation of antimicrobial peptides, proliferation mediators, and adhesion molecules on endothelial cells. In addition, the IL-36-IL-1RL2 axis has an essential role against viral infections, including a potential role in COVID-19 pathology. The evidence presented in this review highlights the importance of the axis IL-36-IL-1RL2 in the development of several inflammation-related diseases and the healing process. It suggests that IL-1RL2 ligands have specific roles depending on the tissue or cell source. However, there is still much to discover about this cytokine family, their functions in other organs, and how they accomplish a dual effect in inflammation and healing.

Mechanism of MyD88S mediated signal termination.

BACKGROUND: A universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)-the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited. METHODS: Luciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD). RESULTS: We show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation). CONCLUSIONS: Our results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction. Video abstract.

Antiviral activity of bacterial TIR domains via immune signalling molecules.

The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems1,2. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule that is a variant of cyclic ADP-ribose3,4. This molecule is hypothesized to mediate plant cell death through a pathway that has yet to be resolved5. TIR domains have also been shown to be involved in a bacterial anti-phage defence system called Thoeris6, but the mechanism of Thoeris defence remained unknown. Here we show that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We also show that, similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe an antiviral signalling pathway in bacteria, and suggest that the generation of intracellular signalling molecules is an ancient immunological function of TIR domains that is conserved in both plant and bacterial immunity.

Activation of TIR signalling boosts pattern-triggered immunity.

Plant immune responses are mainly activated by two types of receptor. Pattern recognition receptors localized on the plasma membrane perceive extracellular microbial features, and nucleotide-binding leucine-rich repeat receptors (NLRs) recognize intracellular effector proteins from pathogens1. NLRs possessing amino-terminal Toll/interleukin-1 receptor (TIR) domains activate defence responses via the NADase activity of the TIR domain2,3. Here we report that activation of TIR signalling has a key role in pattern-triggered immunity (PTI) mediated by pattern recognition receptors. TIR signalling mutants exhibit attenuated PTI responses and decreased resistance against pathogens. Consistently, PTI is compromised in plants with reduced NLR levels. Treatment with the PTI elicitor flg22 or nlp20 rapidly induces many genes encoding TIR-domain-containing proteins, which is likely to be responsible for activating TIR signalling during PTI. Overall, our study reveals that activation of TIR signalling is an important mechanism for boosting plant defence during PTI.

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

Paradoxical Roles of the MAL/Tirap Adaptor in Pathologies.

Toll-like receptors (TLRs) are at the forefront of pathogen recognition ensuring host fitness and eliciting protective cellular and humoral responses. Signaling pathways downstream of TLRs are tightly regulated for preventing collateral damage and loss of tolerance toward commensals. To trigger effective intracellular signaling, these receptors require the involvement of adaptor proteins. Among these, Toll/Interleukin-1 receptor domain containing adaptor protein (Tirap or MAL) plays an important role in establishing immune responses. Loss of function of MAL was associated with either disease susceptibility or resistance. These opposite effects reveal paradoxical functions of MAL and their importance in containing infectious or non-infectious diseases. In this review, we summarize the current knowledge on the signaling pathways involving MAL in different pathologies and their impact on inducing protective or non-protective responses.

Myeloid Differentiation Primary Response Protein 88 (MyD88): The Central Hub of TLR/IL-1R Signaling.

Myeloid differentiation primary response protein 88 (MyD88) is a ubiquitously expressed cytoplasmic adaptor protein that plays a central role in the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways. TLR/IL-1R pathways regulate the proliferation and differentiation of cells involved in the innate and adaptive immunity. Although the general TLR/IL-1R activation cascade is well understood, the molecular mechanisms involving MyD88 have only begun to surface in the past decade. In this review, we explore MyD88 structural biology, the role of posttranslational modifications (PTMs), and the recent developments in MyD88 inhibitor discovery and use. We also highlight the potential application of MyD88-targeted therapies in human diseases.

A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions.

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.

Mining the PDB for Tractable Cases Where X-ray Crystallography Combined with Fragment Screens Can Be Used to Systematically Design Protein-Protein Inhibitors: Two Test Cases Illustrated by IL1ß-IL1R and p38α-TAB1 Complexes.

Nowadays, it is possible to combine X-ray crystallography and fragment screening in a medium throughput fashion to chemically probe the surfaces used by proteins to interact and use the outcome of the screens to systematically design protein-protein inhibitors. To prove it, we first performed a bioinformatics analysis of the Protein Data Bank protein complexes, which revealed over 400 cases where the crystal lattice of the target in the free form is such that large portions of the interacting surfaces are free from lattice contacts and therefore accessible to fragments during soaks. Among the tractable complexes identified, we then performed single fragment crystal screens on two particular interesting cases: the Il1ß-ILR and p38α-TAB1 complexes. The result of the screens showed that fragments tend to bind in clusters, highlighting the small-molecule hotspots on the surface of the target protein. In most of the cases, the hotspots overlapped with the binding sites of the interacting proteins.

Interleukin-1 receptor antagonist, interleukin-2 receptor alpha subunit and amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: To clarify the causal associations of interleukin-1 receptor antagonist (IL-1ra) and interleukin-2 receptor alpha subunit (IL-2rα) with the risk of amyotrophic lateral sclerosis (ALS). METHODS: A two-sample Mendelian randomization study design was employed. Single-nucleotide polymorphisms associated with IL-1ra (n = 2) and IL-2rα (n = 1) at the genome-wide significance level were used as unbiased instrumental variables. Summary-level data for ALS were obtained from Project MinE, an international collaboration consortium with 12 577 ALS cases and 23 475 controls of European descent. RESULTS: Genetic predisposition to higher levels of IL-1ra was significantly associated with lower odds of ALS. For a 1-SD increase of circulating IL-1ra levels, the odds ratio of ALS was 0.64 (95% confidence intervals, 0.46-0.88; P = 0.005). There was a borderline inverse association between IL-2rα levels and ALS (odds ratio, 0.91; 95% confidence intervals, 0.83-1.00; P = 0.058). CONCLUSIONS: Interleukin-1 receptor antagonist levels were inversely associated with ALS, suggesting that interleukin-1 inhibitors may lower the risk of this always fatal disease. The role of IL-2rα levels in ALS needs further verification in causal inference studies with larger sample sizes.

X-ray crystal structure localizes the mechanism of inhibition of an IL-36R antagonist monoclonal antibody to interaction with Ig1 and Ig2 extra cellular domains.

Cellular signaling via binding of the cytokines IL-36α, ß, and γ along with binding of the accessory protein IL-36RAcP, to their cognate receptor IL-36R is believed to play a major role in epithelial and immune cell-mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL-36R and a Fab derived from a high affinity anti-IL-36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL-36R, reveals similarities with other structurally characterized IL-1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL-36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events.

A peptide derived from the core ß-sheet region of TIRAP decoys TLR4 and reduces inflammatory and autoimmune symptoms in murine models.

BACKGROUND: TLRs are some of the actively pursued drug-targets in immune disorders. Owing to a recent surge in the cognizance of TLR structural biology and signalling pathways, numerous therapeutic modulators, ranging from low-molecular-weight organic compounds to polypeptides and nucleic acid agents have been developed. METHODS: A penetratin-conjugated small peptide (TIP3), derived from the core ß-sheet of TIRAP, was evaluated in vitro by monitoring the TLR-mediated cytokine induction and quantifying the protein expression using western blot. The therapeutic potential of TIP3 was further evaluated in TLR-dependent in vivo disease models. FINDINGS: TIP3 blocks the TLR4-mediated cytokine production through both the MyD88- and TRIF-dependent pathways. A similar inhibitory-effect was exhibited for TLR3 but not on other TLRs. A profound therapeutic effect was observed in vivo, where TIP3 successfully alleviated the inflammatory response in mice model of collagen-induced arthritis and ameliorated the disease symptoms in psoriasis and SLE models. INTERPRETATION: Our data suggest that TIP3 may be a potential lead candidate for the development of effective therapeutics against TLR-mediated autoimmune disorders. FUNDING: This work was supported by the National Research Foundation of Korea (NRF-2019M3A9A8065098, 2019M3D1A1078940 and 2019R1A6A1A11051471). The funders did not have any role in the design of the present study, data collection, data analysis, interpretation, or the writing of the manuscript.