1H, 13C, and 15N resonance assignment of the TIR domain of human MyD88 (original) (raw)
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Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling
Proceedings of the National Academy of Sciences, 2009
Myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal) are adaptor molecules critically involved in the Toll-like receptor (TLR) 4 signaling pathway. While Mal has been proposed to serve as a membrane-sorting adaptor, MyD88 mediates signal transduction from activated TLR4 to downstream components. The Toll/Interleukin-1 receptor (TIR) domain of MyD88 is responsible for sorting and signaling via direct or indirect TIR؊TIR interactions between Mal and TLR4. However, the molecular mechanisms involved in multiple interactions of the TIR domain remain unclear. The present study describes the solution structure of the MyD88 TIR domain. Reporter gene assays revealed that 3 discrete surface sites in the TIR domain of MyD88 are important for TLR4 signaling. Two of these sites were shown to mediate direct binding to the TIR domain of Mal. Interestingly, Mal-TIR, but not MyD88-TIR, directly binds to the cytosolic TIR domain of TLR4. These observations suggested that the heteromeric assembly of TIR domains of the receptor and adaptors constitutes the initial step of TLR4 intracellular signal transduction.
Journal of Biological Chemistry, 2009
The adapter protein MyD88 adapter-like (Mal), encoded by TIR-domain containing adapter protein (Tirap) (MIM 606252), is the most polymorphic of the five adapter proteins involved in Toll-like receptor signaling, harboring eight non-synonymous single nucleotide polymorphisms in its coding region. We screened reported mutations of Mal for activity in reporter assays to test the hypothesis that variants of Mal existed with altered signaling potential. A TIR domain variant, Mal D96N (rs8177400), was found to be inactive. In reconstituted cell lines, Mal D96N acted as a hypomorphic mutation, with impaired cytokine production and NF-B activation upon lipopolysaccharide or PAM 2 CSK 4 stimulation. Moreover, co-immunoprecipitation studies revealed that Mal D96N is unable to interact with MyD88, a prerequisite for downstream signaling to occur. Computer modeling data suggested that residue 96 resides in the MyD88 binding site, further supporting these findings. Genotyping of Mal D96N in three different cohorts suggested that it is a rare mutation. We, thus, describe a rare variant in Mal that exerts its effect via its inability to bind MyD88.
Molecular Immunology, 2012
Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligandinduced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR-TIR complexes.
Journal of Biological Chemistry, 2013
Background: MyD88 is an adaptor protein that plays a crucial role in the immune response. Results: We identified residues within the TIR domain of MyD88 required for protein self-association. Conclusion: Interference with the surface of homodimerization identified by these residues inhibits MyD88 function. Significance: The inhibition of MyD88 activity could be a good therapeutic strategy for inflammatory and autoimmune diseases. 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 sitedirected 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 Glu 183 , Ser 244 , and Arg 288 impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-B. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88 151-189 and GFP-MyD88 168-189), comprising the Glu 183 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.
FEBS Letters, 2007
MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.
Binding specificity of Toll-like receptor cytoplasmic domains
European Journal of Immunology, 2006
MyD88 participates in signal transduction by binding to the cytoplasmic Toll/IL-1 receptor (TIR) domains of activated Toll-like receptors (TLR). Yeast two-hybrid experiments reveal that the TIR domains of human TLR differ in their ability to associate with MyD88: The TIR of TLR2 binds to MyD88 but the TIR of the closely related TLR1, 6, or 10 do not. Using chimeric TIR domains, we define the critical region responsible for differential MyD88 binding, and use a computational analysis of the critical region to reveal the amino acids that differ between MyD88 binders and non-binders. Remarkably, a single missense mutation created in TLR1 (N672D) confers on it the ability to bind MyD88, without affecting its association with other proteins. Mutations identified as critical for MyD88 binding also affect signaling of TLR pairs in mammalian cells. To investigate the difference between MyD88 binders and non-binders, we identify novel interacting proteins for each cytoplasmic domain of TLR1, 2, 6, and 10. For example, heat shock protein (HSP)60 binds to TLR1 but not to TLR2, and HSP60 and MyD88 appear to bind the same region of the TIR domain. In summary, interactions between the TLR, MyD88, and novel associated proteins have been characterized. Abbreviations: 3AT: 3-aminotriazole Á AD: Gal4 transcriptional activation domain Á BD: Gal4 DNA-binding domain Á ELAM-1: endothelial cell-leukocyte adhesion molecule-1 Á MAL/ TIRAP: MyD88 adapter-like/TIR domain-containing adapter protein Á TIR: Toll/IL-1 receptor domain Á -TL: lacking tryptophan and leucine Á -TLH: lacking tryptophan, leucine, and histidine Á TRAP: tumor necrosis factor receptor-associated protein Victoria Brown et al.
Journal of Biological Chemistry, 2005
Myeloid differentiation factor MyD88 is the essential adaptor protein that integrates and transduces intracellular signals generated by multiple Toll-like receptors including receptor complex for interleukin (IL) 1, a key inflammatory cytokine. IL1 receptor complex interacts with MyD88 via the Toll/IL1 receptor (TIR) domain. Here we report structure-function studies that help define the MyD88 TIR domain binding sites involved in IL1-induced protein-protein interactions. The MyD88 TIR domain, employed as a dominant negative inhibitor of IL1 signaling to screen MyD88 TIR mutants, lost its suppressing activity upon truncation of its Box 3. Accordingly, mutations of Box 3 residues 285-286 reversed the dominant negative effect of the MyD88 TIR domain on IL1induced and NFB-dependent reporter gene activity and IL6 production. Moreover, mutations of residues 171 in helix ␣A, 195-197 in Box 2, and 275 in E-strand had similar functional effects. Strikingly, only mutations of residues 195-197 eliminated the TIR-TIR interaction of MyD88 and IL1 receptor accessory protein (IL1RAcP), whereas substitution of neighboring canonical Pro 200 by His was without effect. Mutations in Box 2 and 3 prevented homotypic MyD88 oligomerization via TIR domain. Based on this structure-function analysis, a threedimensional docking model of TIR-TIR interaction between MyD88 and IL1RAcP was developed. The importance of the Toll-like receptor (TLR) 1 family in innate immune response to microbial surfaces and nucleic acids is well established. Surprisingly, two members of this family recognize key inflammatory cytokines interleukin (IL) 1 and IL18. These cytokines induce genes that encode other mediators of inflammation such as pleiotropic inflammatory cytokine IL6 and interferon ␥, respectively (1-5).
Journal of Biological …, 2003
The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a ␣--fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.
Proceedings of the National Academy of Sciences, 2011
Initiation of the innate immune response requires agonist recognition by pathogen-recognition receptors such as the Toll-like receptors (TLRs). Toll/interleukin-1 receptor (TIR) domain-containing adaptors are critical in orchestrating the signal transduction pathways after TLR and interleukin-1 receptor activation. Myeloid differentiation primary response gene 88 (MyD88) adaptor-like (MAL)/TIR domain-containing adaptor protein (TIRAP) is involved in bridging MyD88 to TLR2 and TLR4 in response to bacterial infection. Genetic studies have associated a number of unique singlenucleotide polymorphisms in MAL with protection against invasive microbial infection, but a molecular understanding has been hampered by a lack of structural information. The present study describes the crystal structure of MAL TIR domain. Significant structural differences exist in the overall fold of MAL compared with other TIR domain structures: A sequence motif comprising a βstrand in other TIR domains instead corresponds to a long loop, placing the functionally important "BB loop" proline motif in a unique surface position in MAL. The structure suggests possible dimerization and MyD88-interacting interfaces, and we confirm the key interface residues by coimmunoprecipitation using sitedirected mutants. Jointly, our results provide a molecular and structural basis for the role of MAL in TLR signaling and disease protection.
MAPPIT analysis of TLR adaptor complexes
FEBS Letters, 2007
Toll-like receptors (TLRs) are crucial components of the innate immune system, coupling pathogen recognition to a cellular response. We used the MAPPIT mammalian two-hybrid technique to investigate protein-protein interactions in the early steps in TLR signalling. A partial TLR-adaptor interaction map was constructed confirming several known but also documenting novel interactions. We show that the TLR adaptor Mal is critical for linking Myeloid Differentiation primary response protein 88 (MyD88) to TLR2 and TLR4. Analysis of the contributions of the different sub-domains of MyD88-adaptor-like protein (Mal) and MyD88 in adaptor homo-and hetero-dimerisation provides an initial mechanistic insight in this bridging function of Mal.