BB Loop Research Articles

Microcrystal electron diffraction structure of Toll-like receptor 2 TIR-domain-nucleated MyD88 TIR-domain higher-order assembly.

Eukaryotic TIR (Toll/interleukin-1 receptor protein) domains signal via TIR-TIR interactions, either by self-association or by interaction with other TIR domains. In mammals, TIR domains are found in Toll-like receptors (TLRs) and cytoplasmic adaptor proteins involved in pro-inflammatory signaling. Previous work revealed that the MAL TIR domain (MALTIR) nucleates the assembly of MyD88TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88TIR assembly has previously been solved, revealing a two-stranded higher-order assembly of TIR domains. In this work, it is demonstrated that the TIR domain of TLR2, which is reported to signal as a heterodimer with either TLR1 or TLR6, induces the formation of crystalline higher-order assemblies of MyD88TIR in vitro, whereas TLR1TIR and TLR6TIR do not. Using an improved data-collection protocol, the MicroED structure of TLR2TIR-induced MyD88TIR microcrystals was determined at a higher resolution (2.85 Å) and with higher completeness (89%) compared with the previous structure of the MALTIR-induced MyD88TIR assembly. Both assemblies exhibit conformational differences in several areas that are important for signaling (for example the BB loop and CD loop) compared with their monomeric structures. These data suggest that TLR2TIR and MALTIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88TIR assemblies unidirectionally.

Expression of Concern: Divergent Roles of Amino Acid Residues Inside and Outside the BB Loop Affect Human Toll-Like Receptor (TLR)2/2, TLR2/1 and TLR2/6 Responsiveness.

Expression of Concern: Divergent Roles of Amino Acid Residues Inside and Outside the BB Loop Affect Human Toll-Like Receptor (TLR)2/2, TLR2/1 and TLR2/6 Responsiveness.

Structural analysis of the Toll-like receptor 15 TIR domain.

Toll-like receptors (TLRs) activate innate immunity in response to pathogen-associated molecular patterns (PAMPs). The ectodomain of a TLR directly senses a PAMP and the intracellular TIR domain dimerizes to initiate a signaling cascade. The TIR domains of TLR6 and TLR10, which belong to the TLR1 subfamily, have been structurally characterized in a dimer, whereas those of other subfamilies, including TLR15, have not been explored at the structural or molecular level. TLR15 is a TLR unique to birds and reptiles that responds to virulence-associated fungal and bacterial proteases. To reveal how the TLR15 TIR domain (TLR15TIR) triggers signaling, the crystal structure of TLR15TIR was determined in a dimeric form and a mutational study was performed. TLR15TIR forms a one-domain structure in which a five-stranded β-sheet is decorated by α-helices, as shown for TLR1 subfamily members. TLR15TIR exhibits substantial structural differences from other TLRs at the BB and DD loops and αC2 helix that are involved in dimerization. As a result, TLR15TIR is likely to form a dimeric structure that is unique in its intersubunit orientation and the contribution of each dimerizing region. Further comparative analysis of TIR structures and sequences provides insights into the recruitment of a signaling adaptor protein by TLR15TIR.

A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1

Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.

Numerical Simulation of an Out-Vessel Loss of Coolant from the Breeder Primary Loop Due to Large Rupture of Tubes in a Primary Heat Exchanger in the DEMO WCLL Concept

This work presents a thermohydraulic analysis of a postulated accident involving the rupture of the breeder primary cooling loop inside a heat exchanger (once through steam generator). After the detection of the loss of pressure inside the primary loop, a plasma shutdown is actuated with a consequent plasma disruption, isolation of the secondary loop, and shutoff of the pumps in the primary; no other safety counteractions are postulated. The objective of the work is to analyze the pressurization of the primary and secondary sides to show that the accidental overpressure in the two sides of the steam generators is safely accommodated. Furthermore, the effect of the plasma disruption on the FW, in terms of temperatures, should be analyzed. Lastly, the time transients of the pressures and temperatures in the HX and BB for a time span of up to 36 h should be obtained to assess the effect of the decay heat over a long period. A full nodalization of the OTSG was realized together with a simplified nodalization of the whole PHTS BB loop. The code utilized was MELCOR for fusion version 1.8.6. The accident was simulated by activating a flow path which directly connected one section of the primary with the parallel section of the secondary side. It is shown here that the pressures and the temperatures inside the whole PHTS system remain below the safety thresholds for the whole transient.

Use of forearm basilic vein as an option of upper limb vascular access for haemodialysis

Abstract Authors : Mohamed ashraf elsayed elshazli, Samer abdelhamid regal MD , Hossam Mohamed Mustafa MD, Amr Mohamed Mustafa MD . Background: in our stuy , we aimed to evaluate the outcome of forearm basilc vein transposition and anastomosis to either radial or brachial areteries to create arteriovenous fistula for haemodialysis . Patient and Methods : This is an observational prospective study conducted from august 2019 to December 2020 at the Department of Vascular Surgery, Mansoura University hospital . 25 patients were selected for forearm basilic vein transposition (FBVT ) . 18 cases underwemt BB loop AVF and 7 cases underwent radiobasilic AVF . procedure was done under combined supraclavicular and local xylocaine 1% anaethesia . Basilic vein was harvested using multiple incisions then elevated and anastmosed to either radial artery in 7 cases or tunneled as aloop and anastomsed to brachial artery in 18 cases . Results: The mean age of patients who received an AVF was 56.96±9.61 years, with a range of 35 to 70 years, and included 19 men (76%) and 6 women (24%).18 cases underwemt BB looped AVF and 7 cases underwent radiobasilic AVF . patients were followed up for 1 year. 1ry patency rates at 3,6,12 months were 84% ,83.3%,82.6%. Conclusion: Radiobasilic or forearm brachiobasilic looped AVF can be used as an alternative option in case of cephalic vein inadequancy or if it was previously used for haemdialysis.

Structural analysis of toll-like receptor 18 from soiny mullet (Liza haematocheila): Giving insights on the ligand binding mechanism of fish specific TLRs

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of innate immune system, playing crucial roles in immune defense against pathogens. TLR18, a member of TLR1 family, is fish-specific TLR and involves in the immune response against bacterial infection. Currently, the structural biology of fish TLR18 is poorly elaborated. In this study, the structure and ligand binding of TLR18 (smTLR18) of soiny mullet (Liza haematocheila), an economically valuable aquaculture mugilid species, were analyzed. The extracellular domain (ECD) of smTLR18 formed an open-loop horseshoe-shaped structure with the concave surfaces made up of 19 parallel β-strands (LRR1-LRR19), lacking Z-loop that seen in human TLR9. The intracellular Toll/interleukin (IL)-1 (TIR) domain contained a central 4-parallel β-sheet (βA-βD) surrounded by 5 α-helices (αA-αE). Molecular docking analysis revealed that both ECD domain and TIR domain of smTLR18 could form homodimers. For the ECD homodimer, the main residues involved in dimer formation were located from LRR10 to LRR14. For the TIR homodimer, the residues involved in dimer formation were located in BB loop, αB helix, αC helix and DD loop. Ligand binding analyses revealed that peptidoglycans (PGNs) and lipopolysaccharides (LPS), two main bacterial pathogen-associated molecular patterns (PAMPs), were the potential ligands of smTLR18. The van der Waals and Coulombic interactions contributed to the interactions between smTLR18 and PGNs, while only van der Waals dominated the interactions between smTLR18 and LPS. The residues involved in ligands binding were located from LRR9 to LRR13. Our results provided the structural bases for elucidate the ligand binding of fish TLR18.

Insight into the sequence-structure relationship of TLR cytoplasm’s Toll/Interleukin-1 receptor domain towards understanding the conserved functionality of TLR 2 heterodimer in mammals

The signaling response of TLR2 to ligands has always been as a homodimer or in heterodimerization with TLR1/TLR6. The Toll/Interleukin-1 Receptor (TIR) domain of the TLR cytoplasmic region regulates the dimerization and interactions with adaptor molecules to build an active signaling complex. To understand the conservation of functionality of the TLR2-heterodimers between the distantly related species human(h) and mice(m), the pattern of TIR-TIR interaction in heterodimers has been studied through the sequence-structural point of view. Comparative analysis of primary sequence and structural pattern of TLRs(1/2/6) corroborates higher sequence homology between TLR1 and TLR6. Molecular docking analysis of TLR2-TLR1 and TLR2-TLR6 cytoplasmic dimers in both mouse and human have identified that for interaction the BB loop/near-BB loop residues of TLR2 are involved with the near-DD loop of TLR1 and DD loop residues of TLR6 within the TIR domains, which may cause to differential signaling. Molecular dynamics simulation of dimers for both human and mice species recognize stable interface between near-BB/BB loop region of TLR2 and discrete near-DD and DD loop region of TLR1 and TLR6 respectively. The observed dimerization pattern in both the species is further supported by Alanine scanning mutation study. However, Solvent Accessible Surface Area (SASA) of BB and DD loop regions of the cytoplasmic monomers and the heterodimers suggests that while TLR2 BB loop is actively associated as the dimer interface with its heterodimer partners in both the species, the DD loop acts as the active interfacing region in hTLR1 and mTLR6. Communicated by Ramaswamy H. Sarma

A Computational Probe into the Structure and Dynamics of the Full-Length Toll-Like Receptor 3 in a Phospholipid Bilayer.

Toll-like receptor 3 (TLR3) provides the host with antiviral defense by initiating an immune signaling cascade for the production of type I interferons. The X-ray structures of isolated TLR3 ectodomain (ECD) and transmembrane (TM) domains have been reported; however, the structure of a membrane-solvated, full-length receptor remains elusive. We investigated an all-residue TLR3 model embedded inside a phospholipid bilayer using molecular dynamics simulations. The TLR3-ECD exhibited a ~30°–35° tilt on the membrane due to the electrostatic interaction between the N-terminal subdomain and phospholipid headgroups. Although the movement of dsRNA did not affect the dimer integrity of TLR3, its sugar-phosphate backbone was slightly distorted with the orientation of the ECD. TM helices exhibited a noticeable tilt and curvature but maintained a consistent crossing angle, avoiding the hydrophobic mismatch with the bilayer. Residues from the αD helix and the CD and DE loops of the Toll/interleukin-1 receptor (TIR) domains were partially absorbed into the lower leaflet of the bilayer. We found that the previously unknown TLR3-TIR dimerization interface could be stabilized by the reciprocal contact between αC and αD helices of one subunit and the αC helix and the BB loop of the other. Overall, the present study can be helpful to understand the signaling-competent form of TLR3 in physiological environments.

A cell-penetrating peptide blocks Toll-like receptor-mediated downstream signaling and ameliorates autoimmune and inflammatory diseases in mice

Toll-like receptors (TLRs) recognize pathogen/damage-associated molecular patterns and initiate inflammatory signaling cascades. Occasionally, overexpression of TLRs leads to the onset of numerous inflammatory diseases, necessitating the development of selective inhibitors to allow a protective yet balanced immune response. Here, we demonstrate that a novel peptide (TIP1) derived from Toll/interleukin-1 receptor (TIR) domain-containing adapter protein inhibited multiple TLR signaling pathways (MyD88-dependent and MyD88-independent) in murine and human cell lines. TIP1 also inhibited NLRP3-mediated IL-1β secretion, as we validated at both the protein and mRNA levels. Biophysical experiments confirmed that TIP1 specifically binds to the BB loop of the TLR4-TIR domain. Animal studies revealed that TIP1 inhibited the secretion of lipopolysaccharide (LPS)-induced proinflammatory cytokines in collagen-induced arthritis (CIA) and kaolin/carrageenan-induced arthritis (K/C) rodent models. TIP1 also rescued animals from sepsis and from LPS-induced kidney/liver damage. Importantly, TIP1 ameliorated the symptoms of rheumatoid arthritis in CIA and K/C rodent models, suggesting that TIP1 has therapeutic potential for the treatment of TLR-mediated autoimmune/inflammatory diseases.

Blocking TIR Domain Interactions in TLR9 Signaling.

Interaction of TLR9 with ligands activates NF-κB, leading to proinflammatory cytokine production. Excessive TLR activation is a pathogenic factor for inflammatory diseases. This study has examined cell-permeating decoy peptides (CPDPs) derived from the TLR9 Toll/IL-1R resistance (TIR) domain. CPDP 9R34, which included AB loop, β-strand B, and N-terminal BB loop residues, inhibited TLR9 signaling most potently. CPDPs derived from α-helices C, D, and E (i.e., 9R6, 9R9, and 9R11) also inhibited TLR9-induced cytokines but were less potent than 9R34. 9R34 did not inhibit TLR2/1, TLR4, or TLR7 signaling. The N-terminal deletion modification of 9R34, 9R34-ΔN, inhibited TLR9 as potently as the full length 9R34. Binding of 9R34-ΔN to TIR domains was studied using cell-based Förster resonance energy transfer/fluorescence lifetime imaging approach. Cy3-labeled 9R34-ΔN dose-dependently decreased fluorescence lifetime of TLR9 TIR-Cerulean (Cer) fusion protein. Cy3-9R34-ΔN also bound TIRAP TIR, albeit with a lesser affinity, but not MyD88 TIR, whereas CPDP from the opposite TIR surface, 9R11, bound both adapters and TLR9. i.p. administration of 9R34-ΔN suppressed oligonucleotide-induced systemic cytokines and lethality in mice. This study identifies a potent, TLR9-specific CPDP that targets both receptor dimerization and adapter recruitment. Location of TIR segments that represent inhibitory CPDPs suggests that TIR domains of TLRs and TLR adapters interact through structurally homologous surfaces within primary receptor complex, leading to formation of a double-stranded, filamentous structure. In the presence of TIRAP and MyD88, primary complex can elongate bidirectionally, from two opposite ends, whereas in TIRAP-deficient cells, elongation is unidirectional, only through the αE side.

Selective disruption of TLR2-MyD88 interaction inhibits inflammation and attenuates Alzheimer's pathology.

Induction of TLR2 activation depends on its association with the adapter protein MyD88. We have found that TLR2 and MyD88 levels are elevated in the hippocampus and cortex of patients with Alzheimer’s disease (AD) and in a 5XFAD mouse model of AD. Since there is no specific inhibitor of TLR2, to target induced TLR2 from a therapeutic angle, we engineered a peptide corresponding to the TLR2-interacting domain of MyD88 (TIDM) that binds to the BB loop of only TLR2, and not other TLRs. Interestingly, WT TIDM peptide inhibited microglial activation induced by fibrillar Aβ1-42 and lipoteichoic acid, but not 1-methyl-4-phenylpyridinium, dsRNA, bacterial lipopolysaccharide, flagellin, or CpG DNA. After intranasal administration, WT TIDM peptide reached the hippocampus, reduced hippocampal glial activation, lowered Aβ burden, attenuated neuronal apoptosis, and improved memory and learning in 5XFAD mice. However, WT TIDM peptide was not effective in 5XFAD mice lacking TLR2. In addition to its effects in 5XFAD mice, WT TIDM peptide also suppressed the disease process in mice with experimental allergic encephalomyelitis and collagen-induced arthritis. Therefore, selective targeting of the activated status of 1 component of the innate immune system by WT TIDM peptide may be beneficial in AD as well as other disorders in which TLR2/MyD88 signaling plays a role in disease pathogenesis.

Development of a Novel Backbone Cyclic Peptide Inhibitor of the Innate Immune TLR/IL1R Signaling Protein MyD88

MyD88 is a cytoplasmic adaptor protein that plays a central role in signaling downstream of the TLRs and the IL1R superfamily. We previously demonstrated that MyD88 plays a critical role in EAE, the murine model of multiple sclerosis, and showed that the MyD88 BB-loop decoy peptide RDVLPGT ameliorates EAE. We now designed and screened a library of backbone cyclized peptides based on the linear BB loop peptide, to identify a metabolically stable inhibitor of MyD88 that retains the binding properties of the linear peptide. We identified a novel cyclic peptide protein mimetic that inhibits inflammatory responses to TLR ligands, and NFκB activation in response to IL-1 activation. The inhibitor, c(MyD 4-4), is metabolically stable in comparison to the linear peptide, blocks MyD88 in a specific manner, and inhibits MyD88 function by preventing MyD88 dimerization. Finally, treatment of mice with c(MyD 4-4) reduced the severity of clinical disease in the murine EAE model of multiple sclerosis. Thus, modulation of MyD88-dependent signaling using c(MyD 4-4) is a potential therapeutic strategy to lower innate immune inflammation in autoimmune CNS disease.

Identifying the inhibition of TIR proteins involved in TLR signalling as an anti-inflammatory strategy

Toll/IL1 receptor (TIR) adaptor proteins continue to be an integral part of Toll-like receptors’ (TLR) signalling involved in inflammation. Signalling is likely to be initiated by these TIR adaptors when they are recruited to a TIR–TIR interface formed by TLR dimerization. Among these, myeloid differentiation factor-88 (MyD88), MyD88 adapter-like protein (Mal), TIR domain-containing adaptor protein inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) play pivotal roles at many steps in the signalling events leading to inflammation. The presence of the conserved BB loop residues in the TIR domain of all these important adaptor proteins make them possible targets for inhibition by synthetic compounds. We have designed compounds based on an already known MyD88 TIR dimerization inhibitor, T6167923, which binds well not only to the original target but also to the TIR domains of Mal, TRIF and TRAM. The designed inhibitors are based on modifications of the bromophenyl-sulphonyl-thiophenyl-piperazine-carboxamide series of compounds. We have further suggested modifications in these high-affinity compounds for efficient absorption inside the body. Further, a pharmacophore model highlighting important structural interaction features has been developed. The screened compounds are better in binding to the TIR proteins then the parent compound and hence are good starting points for multi-TIR inhibition.

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 (Lpsd 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.

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.

The evolutionary characteristics and structural biology of Gallus toll-like receptor 21.

Toll‐like receptors (TLRs) are an important part of the innate immune system, acting as a first line of defense against many invading pathogens. The ligand known to bind Gallus toll‐like receptor 21 (gTLR21) is the unmethylated cytosine phosphate guanine dideoxy nucleotide motif; however, the evolutionary characteristics and structural biology of gTLR21 are poorly elaborated. Our results suggest that gTLR21 is phylogenetically and evolutionarily related to the TLR11 family and is perhaps a close ortholog of the Mus TLR13. Structural biology of homology modeling of the gTLR21 ectodomain structure suggests that it has no Z‐loop like that seen in Mus TLR9. The cytosolic toll‐IL‐1 receptor region of gTLR21 contains a central 4‐stranded parallel β‐sheet (βA‐βD) surrounded by 5 α‐helices (αA‐αE) on both sides, a highly conserved structure also seen in other TLRs. Molecular docking analysis reveals that the gTLR21 ectodomain has the potential to distinguish between different ligands. Homodimer analysis results also suggest that Phe842 and Pro844 of the BB loop and Cys876 of the αC helix in gTLR21 are conserved in other cytosolic toll‐IL‐1 receptor domains of other TLRs and may contribute to the docking of homodimers. Our study on the evolutionary characteristics and structural biology of gTLR21 reveals that the molecule may have a broader role to play in innate immune system; however, further experimental validation is required to confirm our findings.

Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling

MyD88 adaptor-like (MAL) is a critical protein in innate immunity, involved in signaling by several Toll-like receptors (TLRs), key pattern recognition receptors (PRRs). Crystal structures of MAL revealed a nontypical Toll/interleukin-1 receptor (TIR)-domain fold stabilized by two disulfide bridges. We therefore undertook a structural and functional analysis of the role of reactive cysteine residues in the protein. Under reducing conditions, the cysteines do not form disulfides, but under oxidizing conditions they are highly amenable to modification. The solution structure of the reduced form of the MAL TIR domain, determined by NMR spectroscopy, reveals a remarkable structural rearrangement compared with the disulfide-bonded structure, which includes the relocation of a β-strand and repositioning of the functionally important "BB-loop" region to a location more typical for TIR domains. Redox measurements by NMR further reveal that C91 has the highest redox potential of all cysteines in MAL. Indeed, mass spectrometry revealed that C91 undergoes glutathionylation in macrophages activated with the TLR4 ligand lipopolysaccharide (LPS). The C91A mutation limits MAL glutathionylation and acts as a dominant negative, blocking the interaction of MAL with its downstream target MyD88. The H92P mutation mimics the dominant-negative effects of the C91A mutation, presumably by preventing C91 glutathionylation. The MAL C91A and H92P mutants also display diminished degradation and interaction with interleukin-1 receptor-associated kinase 4 (IRAK4). We conclude that in the cell, MAL is not disulfide-bonded and requires glutathionylation of C91 for signaling.

Integrative modelling of TIR domain-containing adaptor molecule inducing interferon-\u03b2 (TRIF) provides insights into its autoinhibited state

BackgroundTRIF is a key protein in antiviral innate immunity, operating downstream of TLRs. TRIF activation leads to the production of interferon-β and pro-inflammatory cytokines. There is evidence from experiments to suggest that the N-terminal domain of TRIF binds to its TIR domain to avoid constitutive activation. However, no structure of a complex between the N-terminal domain and the TIR domain exists till date. The disordered nature of the region connecting the N-terminal domain and the TIR domain compounds the issue of elucidating the mechanism of autoinhibition of TRIF. In this study, we have employed an integrative approach consisting of mutual information analysis, docking, molecular dynamics simulations and residue network analysis, in combination with existing experimental data to provide a glimpse of TRIF in its autoinhibited state.ResultsOur extensive docking approach reveals that the N-terminal domain binds to the BB loop-B helix region of the TIR domain, consistent with experimental observations. Long length molecular dynamics simulations of 1 microsecond performed on the docked model highlights residues participating in hydrogen bonding and hydrophobic interactions at the interface. A pair of residues present in the vicinity of the interface is also predicted by mutual information analysis, to co-evolve. Residues mediating long-range interactions within the TIR domain of TRIF were identified using residue network analysis.ConclusionsBased on the results of the modelling and residue network analysis, we propose that the N-terminal domain binds to the BB loop region of the TIR domain, thereby preventing its homodimersation. The binding of TRIF to TLR3 or TRAM could induce a slight conformational change, causing the interactions between the N-terminal domain and TIR domain to disrupt, thereby exposing the BB loop and rendering it amenable for higher-order oligomerisation.ReviewersThis article was reviewed by Michael Gromiha, Srikrishna Subramaniam and Peter Bond (nominated by Chandra Verma).

SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury-induced SARM1 activation

Axon injury in response to trauma or disease stimulates a self-destruction program that promotes the localized clearance of damaged axon segments. Sterile alpha and Toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is an evolutionarily conserved executioner of this degeneration cascade, also known as Wallerian degeneration; however, the mechanism of SARM1-dependent neuronal destruction is still obscure. SARM1 possesses a TIR domain that is necessary for SARM1 activity. In other proteins, dimerized TIR domains serve as scaffolds for innate immune signaling. In contrast, dimerization of the SARM1 TIR domain promotes consumption of the essential metabolite NAD+ and induces neuronal destruction. This activity is unique to the SARM1 TIR domain, yet the structural elements that enable this activity are unknown. In this study, we identify fundamental properties of the SARM1 TIR domain that promote NAD+ loss and axon degeneration. Dimerization of the TIR domain from the Caenorhabditis elegans SARM1 ortholog TIR-1 leads to NAD+ loss and neuronal death, indicating these activities are an evolutionarily conserved feature of SARM1 function. Detailed analysis of sequence homology identifies canonical TIR motifs as well as a SARM1-specific (SS) loop that are required for NAD+ loss and axon degeneration. Furthermore, we identify a residue in the SARM1 BB loop that is dispensable for TIR activity yet required for injury-induced activation of full-length SARM1, suggesting that SARM1 function requires multidomain interactions. Indeed, we identify a physical interaction between the autoinhibitory N terminus and the TIR domain of SARM1, revealing a previously unrecognized direct connection between these domains that we propose mediates autoinhibition and activation upon injury.