An extensively associated dimer in the structure of the C713S mutant of the TIR domain of human TLR2

doi:10.1016/S0006-291X(02)02581-0

Biochemical and Biophysical Research Communications

Volume 299, Issue 2, 29 November 2002, Pages 216-221

https://doi.org/10.1016/S0006-291X(02)02581-0 Get rights and content

Abstract

The Toll/interleukin-1 receptor (TIR) domains are conserved modules in the intracellular regions of the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). The domains are crucial for the signal transduction by these receptors, through homotypic interactions among the receptor and the downstream adapter TIR domains. Previous studies showed that the BB loop in the structure of the TIR domain forms a prominent conserved feature on the surface and is important for receptor signaling. Here we report the crystal structure of the C713S mutant of the TIR domain of human TLR2. An extensively associated dimer is observed in the crystal structure and mutations of several residues in this dimer interface abolished the function of the receptor. Moreover, the structure shows that the BB loop can adopt different conformations, which are required for the formation of this dimer. This asymmetric dimer might represent the TLR2:TLRx heterodimer in the function of this receptor.

Section snippets

Materials and methods

Cloning, protein expression, and purification. The C713S mutant of the TIR domain was prepared with the QuikChange mutagenesis kit (Stratagene) and sequenced to confirm the incorporation of the mutation. The mutant protein was expressed and purified using the same protocol as that for the wild-type protein [13].

Crystallization and data collection. Crystals of the C713S mutant were obtained at 21 °C by the hanging drop vapor diffusion method. The reservoir solution contained 100 mM MES (pH 6.5) or

Overall structure and conformational variability of the BB loop

The crystal structure of the C713S mutant of the TIR domain of human TLR2 has been determined at 3.2 Å resolution. The statistics for the structure refinement are summarized in Table 1. The atomic structure has been deposited at the Protein Data Bank (Accession No. 1077). There are five copies of the TIR domain molecule in the crystallographic asymmetric unit and they will be called molecules A through E here. In all these molecules, the first 13 residues of the protein (626–638) and a portion

Acknowledgements

We thank Kevin D’Amico and Steve Wassermann for access to the 32-ID beamline, Craig Ogata for access to the X4A beamline, Ming Li for helping to set up the TLR2 receptor assays, and John Sims for helpful discussions. This research was supported in part by a grant from the National Institutes of Health (AI49475 to L.T.).

References (22)

J.A. Hoffmann et al.
Phylogenetic perspectives in innate immunity
Science
(1999)
R. Medzhitov et al.
Innate immunity: the virtues of a nonclonal system of recognition
Cell
(1997)
A. Aderem et al.
Toll-like receptors in the induction of the innate immune response
Nature
(2000)
S. Akira et al.
Toll-like receptors: critical proteins linking innate and acquired immunity
Nat. Immunol.
(2001)
D.M. Underhill et al.
Toll-like receptors: key mediators of microbe detection
Curr. Opin. Immunol.
(2002)
N.J. Gay et al.
Drosophila Toll and IL-1 receptor
Nature
(1991)
D.S. Schneider et al.
Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal–ventral polarity in the Drosophila embryo
Genes Dev.
(1991)
A. Poltorak et al.
Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene
Science
(1998)
A.O. Aliprantis et al.
Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2
Science
(1999)
E. Lien et al.
Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products
J. Biol. Chem.
(1999)

O. Takeuchi et al.

Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components

Immunity

(1999)

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The cytoplasmic Toll/interleukin-1 receptor (TIR) domains of IL-1 receptors (IL-1Rs) are evolutionally conserved and essential for transmitting signals. IL-1RAcP is a shared co-receptor in the IL-1R family for signaling. Its splicing form IL-1RAcPb contains a different TIR domain and is unable to transduce NF-κB signaling. Here, we determined crystal structures of TIR domains of IL-1RAcPb and other IL-1Rs including IL-18Rβ, IL-1RAPL2, and zebrafish SIGIRR (zSIGIRR). Structurally variant regions in the TIR domain important for signaling were revealed by structural comparisons. Taking advantage of the IL-1RAcP/IL-1RAcPb pair, we demonstrated that differential TIR domain determines signaling discrepancies between IL-1RAcP and IL-1RAcPb. We also proved the functional importance of two helices (αC and αD) in the structurally variable regions and pinpointed critical residues in αC and αD for signaling. These results collectively provide additional and important knowledge for fully understanding the molecular basis of IL-1R TIR domain in mediating signaling.
Characterization, expression and function analysis of the TLR3 gene in golden pompano (Trachinotus ovatus)
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Toll-like receptors (TLRs)are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many fishes, Toll-like receptors subfamily contain a large number of members with different functions that need to research in deep. In the present study, the full-length cDNA of TLR3 from the golden pompano, Trachinotus ovatus, was cloned and characterized. The full length of ToTLR3 cDNA was 3710 bp including an open reading frame of 2760 bp encoding a peptide of 919 amino acids. The derived amino acids sequence comprised of 14 leucine-rich repeats (LRR), capped with LRRCT followed by transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). Multiple sequence alignment and phylogenetic analysis revealed that ToTLR3 shared the highest similarity to the teleost fish and suggested ToTLR3 is fairly conservative in evolution process. Tissues distribution analysis indicated that ToTLR3 showed a tissue-specific variation with high expression in blood and liver. After the fish were stimulated by poly(I:C), flagellin and LPS, ToTLR3 expression in the liver, intestine, blood, kidney, skin and muscle was significantly upregulated in a time-depended manner, especially in immune related tissues such as liver, blood and kidney. Binding assay revealed the specificity of rToTLR3 for pathogen-associated molecular patterns (PAMPs) and bacteria that included Vibrio harveyi, V. vulnificus, V. anguillarum, Photobacterium damselae, Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus and PolyI:C, LPS, Flagellin, and PGN. In addition, a luciferase reporter assay showed that overexpression ToTLR3 significantly increased NF-κB activity. Collectively, our results suggested that ToTLR3 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, and transmiss the danger signal to downstream signaling pathways.
Regulation of signaling by cooperative assembly formation in mammalian innate immunity signalosomes by molecular mimics
2020, Seminars in Cell and Developmental Biology
Innate immunity pathways constitute the first line of defense against infections and cellular damage. An emerging concept in these pathways is that signaling involves the formation of finite (e.g. rings in NLRs) or open-ended higher-order assemblies (e.g. filamentous assemblies by members of the death-fold family and TIR domains). This signaling by cooperative assembly formation (SCAF) mechanism allows rapid and strongly amplified responses to minute amounts of stimulus. While the characterization of the molecular mechanisms of SCAF has seen rapid progress, little is known about its regulation. One emerging theme involves proteins produced both in host cells and by pathogens that appear to mimic the signaling components. Recently characterized examples involve the capping of the filamentous assemblies formed by caspase-1 CARDs by the CARD-only protein INCA, and those formed by caspase-8 by the DED-containing protein MC159. By contrast, the CARD-only protein ICEBERG and the DED-containing protein cFLIP incorporate into signaling filaments and presumably interfere with proximity based activation of caspases. We review selected examples of SCAF in innate immunity pathways and focus on the current knowledge on signaling component mimics produced by mammalian and pathogen cells and what is known about their mechanisms of action.
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Citation Excerpt :
Both SEFIR family and TIR family belong to the STIR superfamily, of which the members share similar α/β fold and are known to mediate physical protein-protein interactions in cytosolic signal transductions. Several TIR-TIR complex structures have been reported, including human, plant and microbial TIR domains (e.g. TLR2, TLR6, TLR10, MAL/TIRAP, IL-1RAPL, L6, RPS4, RRS1TcpB, PdTIR etc.) (Tao et al., 2002; Valkov et al., 2011; Lin et al., 2012; Nyman et al., 2008; Alaidarous et al., 2014; Jang and Park, 2014; Chan et al., 2010; Bernoux et al., 2011; Snyder et al., 2014; Khan et al., 2004). Structural analysis combined with biochemical data revealed that TIR domain forms various different kinds of dimers.
SEFIR domain-containing proteins are crucial for mammalian adaptive immunity. As a unique intracellular signaling domain, the SEFIR-SEFIR interactions mediate physical protein-protein interactions in the immune signaling network, especially the IL-17- and IL-25-mediated pathways. However, due to the lack of structural information, the detailed molecular mechanism for SEFIR-SEFIR assembly remains unclear. In the present study, we solved the crystal structures of a prokaryotic SEFIR domain from Bacillus cereus F65185 (BcSEFIR), where the SEFIR domain is located at the N terminus. The structure of BcSEFIR revealed two radically distinct SEFIR-SEFIR interaction modes. In the asymmetric form, the C-terminal tail of one SEFIR binds to the helix αA and βB–αB′ segment of the other one, while in the symmetric form, the helices ηC and αE and the DE-segment compose the inter-protomer interface. The C-terminal tail of BcSEFIR, critical for asymmetric interaction, is highly conserved among the SEFIR domains of Act1 orthologs from different species, in particular three absolutely conserved residues that constitute an EXXXXPP motif. In the symmetric interaction mode, the most significant contacts made by residues on helix αE are highly conserved in Act1 SEFIR domains, constituted an RLI/LXE motif. The two novel SEFIR-SEFIR interaction modes might explain the structural basis for SEFIR domain-mediated complex assembly in signaling pathways.
A polar SxxS motif drives assembly of the transmembrane domains of Toll-like receptor 4
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Like all members of the Toll-like receptor (TLR) family, TLR4 comprises of a large ectodomain (ECD) involved in ligand recognition at the cell-surface, and a cytosolic Toll/interleukin-1 receptor (TIR) signalling domain, linked by a lipid membrane-anchored transmembrane (TM) domain (TMD). Binding of immunostimulatory pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide (LPS) to myeloid differentiation factor 2 (MD-2) coreceptor-complexed TLR4 leads to its dimerization, resulting in productive juxtaposition of TIR domains and the initiation of pro-inflammatory innate immune responses. Whilst the process of PAMP recognition is relatively well understood, thanks to numerous high-resolution crystallographic structures of ECDs, the mechanism by which such recognition is translated into TMD dimerization and activating conformational changes is less clear. Based on available biophysical and biochemical experimental data, ab initio modelling, and multiscale molecular dynamics (MD) simulations entailing a total of > 13 μs and > 200 μs of atomistic and coarse-grained sampling, respectively, we investigate the structural basis for TLR4 TMD dimerization within a biologically relevant lipid membrane environment. A key polar-xx-polar (⁶³⁷SxxS⁶⁴⁰) motif is shown to drive association of the TLR4 TMDs, and to maintain a flexible interface, which may be disrupted by selected point mutations. Furthermore, MD simulations of various TMD + ECD constructs have been used to investigate the coupling between domains, revealing that flexible linkers abrogate dimerization via aggregation of ECDs at the membrane surface, explaining previous biochemical observations. These results improve our understanding of the assembly and signalling mechanisms of TLR4, and pave the way for rational structure-based development of membrane-associated immunomodulatory molecules.
The molecular mechanisms of signaling by cooperative assembly formation in innate immunity pathways
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The innate immune system is the first line of defense against infection and responses are initiated by pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs). PRRs also detect endogenous danger-associated molecular patterns (DAMPs) that are released by damaged or dying cells. The major PRRs include the Toll-like receptor (TLR) family members, the nucleotide binding and oligomerization domain, leucine-rich repeat containing (NLR) family, the PYHIN (ALR) family, the RIG-1-like receptors (RLRs), C-type lectin receptors (CLRs) and the oligoadenylate synthase (OAS)-like receptors and the related protein cyclic GMP-AMP synthase (cGAS). The different PRRs activate specific signaling pathways to collectively elicit responses including the induction of cytokine expression, processing of pro-inflammatory cytokines and cell-death responses. These responses control a pathogenic infection, initiate tissue repair and stimulate the adaptive immune system. A central theme of many innate immune signaling pathways is the clustering of activated PRRs followed by sequential recruitment and oligomerization of adaptors and downstream effector enzymes, to form higher-order arrangements that amplify the response and provide a scaffold for proximity-induced activation of the effector enzymes. Underlying the formation of these complexes are co-operative assembly mechanisms, whereby association of preceding components increases the affinity for downstream components. This ensures a rapid immune response to a low-level stimulus. Structural and biochemical studies have given key insights into the assembly of these complexes. Here we review the current understanding of assembly of immune signaling complexes, including inflammasomes initiated by NLR and PYHIN receptors, the myddosomes initiated by TLRs, and the MAVS CARD filament initiated by RIG-1. We highlight the co-operative assembly mechanisms during assembly of each of these complexes.

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¹: Present address: Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Overall structure and conformational variability of the BB loop

Acknowledgements

References (22)

Phylogenetic perspectives in innate immunity

Science

Innate immunity: the virtues of a nonclonal system of recognition

Cell

Toll-like receptors in the induction of the innate immune response

Nature

Toll-like receptors: critical proteins linking innate and acquired immunity

Nat. Immunol.

Toll-like receptors: key mediators of microbe detection

Curr. Opin. Immunol.

Drosophila Toll and IL-1 receptor

Nature

Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal–ventral polarity in the Drosophila embryo

Genes Dev.

Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene

Science

Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2

Science

Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products

J. Biol. Chem.

Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components

Immunity

Cited by (99)

Structural basis of the IL-1 receptor TIR domain-mediated IL-1 signaling

Characterization, expression and function analysis of the TLR3 gene in golden pompano (Trachinotus ovatus)

Regulation of signaling by cooperative assembly formation in mammalian innate immunity signalosomes by molecular mimics

Structure of a prokaryotic SEFIR domain reveals two novel SEFIR-SEFIR interaction modes

A polar SxxS motif drives assembly of the transmembrane domains of Toll-like receptor 4

The molecular mechanisms of signaling by cooperative assembly formation in innate immunity pathways