Structural and Functional Evidence for the Role of the TLR2 DD Loop in TLR1/TLR2 Heterodimerization and Signaling

Gautam, Jitendra; Ashish, .; Comeau, Laurey; Krueger, Joanna K.; Smith, Michael F.

doi:10.1074/jbc.m602057200

Cited by 105 publications

(108 citation statements)

References 32 publications

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“…The highly flexible BB loop is central to many molecular interactions involving TIR domains and adopts different conformations as observed in functional and structural studies of TLR1, TLR2, TLR10, and, recently, TLR6 (26,31,(35)(36)(37). Homodimeric molecular interactions observed in structural studies of hTLR1, hTLR2, and hTLR10 are largely mediated by residues found on the BB loop, DD loop, and α-C helix (26,31,36,37) (Fig.…”

Section: C29 Blocks Tlr2 Bacterial Agonist-induced Proinflammatory Genementioning

confidence: 99%

“…Alanine scanning mutagenesis of all 10 BB loop pocket residues, as well as three additional mutations (Y641A, P681H, and Q747A) was performed, and their effect on TLR2/1 and TLR2/6 signaling in the absence or presence of C29 was examined using our NF-κB reporter assay. Y641A and P681H were shown previously to play a role in TLR2-MyD88 interaction (20,26), and Gautam et al (31) reported that Q747 was not important for TLR2 signaling, and therefore served as a control mutation. All 10 BB loop pocket mutants, including Y647A and P681H, were critical for TLR2/1 signaling (Fig.…”

Section: C29 Blocks Tlr2 Bacterial Agonist-induced Proinflammatory Genementioning

confidence: 99%

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Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Mistry¹,

Laird²,

Schwarz³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

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Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C 16 H 15 NO 4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.small molecule inhibitor | BB loop | TLR2 pocket | CADD T oll-like receptors (TLRs) are type I transmembrane receptors that detect conserved "pathogen-associated molecular patterns" from microbes, as well as host-derived "danger-associated molecular patterns" (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4-9).Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (i.e., nuclear translocation of NF-κB, activation of MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in numerous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, ischemia/reperfusion injury) (11)(12)(13)(14). Several inhibitors of TLR2 signaling have been developed (15-18), yet none is licensed for human use. A better understanding of the Toll/IL-1 receptor resistance (TIR) domain interactions involved in TLR2 signaling could lead to novel therapeutic agents.Both TLRs and adapter proteins contain a cytoplasmic TIR domain that mediates homotypic and heterotypic interactions ...

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Section: C29 Blocks Tlr2 Bacterial Agonist-induced Proinflammatory Genementioning

confidence: 99%

Section: C29 Blocks Tlr2 Bacterial Agonist-induced Proinflammatory Genementioning

confidence: 99%

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Mistry¹,

Laird²,

Schwarz³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

View full text Add to dashboard Cite

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“…It has been shown that during these interactions, the TIR domains react with each other physically [61]. Furthermore, TIR domains condition the heterodimerization of some animal TLR receptors [62]. An analogous role of the TIR domain has been proposed for plants.…”

Section: Other Domainsmentioning

confidence: 99%

R proteins as fundamentals of plant innate immunity

Głowacki

Macioszek

Kononowicz

2011

Cellular and Molecular Biology Letters

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Abstract:Plants are attacked by a wide spectrum of pathogens, being the targets of viruses, bacteria, fungi, protozoa, nematodes and insects. Over the course of their evolution, plants have developed numerous defense mechanisms including the chemical and physical barriers that are constitutive elements of plant cell responses locally and/or systemically. However, the modern approach in plant sciences focuses on the evolution and role of plant protein receptors corresponding to specific pathogen effectors. The recognition of an invader's molecules could be in most cases a prerequisite sine qua non for plant survival. Although the predicted three-dimensional structure of plant resistance proteins (R) is based on research on their animal homologs, advanced technologies in molecular biology and bioinformatics tools enable the investigation or prediction of interaction mechanisms for specific receptors with pathogen effectors. Most of the identified R proteins belong to the NBS-LRR family. The presence of other domains (including the TIR domain) apart from NBS and LRR is fundamental for the classification of R proteins into subclasses. Recently discovered additional domains (e.g. WRKY) of R proteins allowed the examination of their localization in plant cells and the role they play in signal transduction during the plant resistance response to biotic stress factors. This review focuses on the current state of knowledge about the NBS-LRR family of plant R proteins: their structure, function and evolution, and the role they play in plant innate immunity.

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“…Despite extensive structural studies, it is not known why homotypic interactions are essential for downstream signaling (20)(21)(22)(23)(24)(25)(26)(27). To address this issue, it is necessary to discriminate residues required for homotypic and those required for heterotypic interactions.…”

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confidence: 99%

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling

Enokizono

Kumeta

Funami

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling. A large number of TIR domain structures, including receptors and adaptors, have been determined by X-ray crystallography and NMR. The receptors include TLR1 (9), TLR2 (10), and IL-1R accessory protein-like (IL-1RAPL) (11). Adaptors include myeloid differentiation factor 88 (MyD88) (12) and MyD88 adaptorlike (Mal) (13,14). In addition, AtTIR (15, 16) derived from Arabidopsis thaliana and PdTIR (17) from bacteria have been solved. Each of these TIR domain structures has a ferredoxin fold with five β-strands (βA-βE), five α-helices (αA-αE), and loops connecting β-strands and α-helices (9). Although homotypic interactions of the TIR domains have been proposed based on the crystal structures, most proposed models have small interacting surfaces, possibly due to crystal contacts. Recently, however, a crystal structure of the TLR10 TIR domain was reported that forms a homotypic dimer mediated by the loop connecting βB and αB (designated "BB-loop") (18). Interestingly, BB-loop mutations in TLR4 were reported to be dominant-negative and abrogated downstream signaling (19). TICAM-1 and TICAM-2 harboring BB-loop mutations are also dominant-negative and unable to form homotypic interactions (1, 2), reinforcing the importance of BB-loop-mediated homotypic dimer formation in signal propagation.Despite extensive structural studies, it is not known why homotypic interactions are essential for downstream signaling (20-27). To address this issue, it is necessary to discriminate residues required for homotypic and those required for heterotypic interactions. Here, we first determine the structures of the monomeric BB-loop mutants of the TICAM-1 and TICAM-2 TIR domains using NMR. Then, based on the solution structures of the BB-loop mutants, coupled mutagenesis/yeast two-hybrid experiments, and restrained docking calculations, we show that the homotypic interaction of TICAM-2 TIR is es...

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Structural and Functional Evidence for the Role of the TLR2 DD Loop in TLR1/TLR2 Heterodimerization and Signaling

Cited by 105 publications

References 32 publications

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

R proteins as fundamentals of plant innate immunity

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling

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