Structural Complementarity of Toll/Interleukin-1 Receptor Domains in Toll-like Receptors and the Adaptors Mal and MyD88

112

102

The Toll family of receptors is required for innate immune response to pathogen-associated molecules, but the mechanism of signaling is not entirely clear. In Drosophila the prototypic Toll regulates both embryonic development and adult immune response. We demonstrate here that the host protein Spä tzle can function as a ligand for Toll because Spä tzle forms a complex with Toll in transgenic fly extracts and stimulates the expression of a Tolldependent immunity gene, drosomycin, in adult flies. We also show that constitutively active mutants of Toll form multimers that contain intermolecular disulfide linkages. These disulfide linkages are critical for the activity of one of these mutant receptors, indicating that multimerization is essential for the constitutive activity. Furthermore, systematic mutational analysis revealed that a conserved cysteine-containing motif, different from the cysteines used for the intermolecular disulfide linkages, serves as a selfinhibitory module of Toll. Deleting or mutating this cysteinecontaining motif leads to constitutive activity. This motif is located just outside the transmembrane domain and may provide a structural hindrance for multimerization and activation of Toll. Together, our results suggest that multimerization may be a regulated, essential step for Toll-receptor activation.U pon infection, insects mount a rapid but rather nonspecific response. This insect antimicrobial response is similar to the mammalian innate immune response (1-7). The recognition receptors of the innate immune system have limited diversity and are designed to recognize pathogen-associated molecules such as lipopolysaccharides and peptidoglycans, which are common molecules found in groups of microorganisms. Toll and Toll-like receptors (TLRs) are a conserved family of recognition receptors in insects and mammals (5, 6). The Toll family of receptors is essential for innate immunity, but it is unclear how the receptors interact with microbial substances and host ligands.In Drosophila, the prototypic Toll receptor was first identified as an essential component for embryonic development (8). Subsequently, Toll was also shown to be essential for the induced expression of a subset of antimicrobial peptides during antifungal and anti-Gram-positive bacterial responses (9, 10). The regulation of the anti-Gram-negative bacterial response depends on a second pathway called the Immune deficiency (Imd) pathway, which controls the expression of another subset of antimicrobial peptides (2-4, 11). The induced expression of antimicrobial peptides through these two pathways is critical for Drosophila to survive microbial infections (12).The mechanism by which Toll receptors transmit the signal of infection across the cell membrane is not clear. Many components of the Toll pathway in the Drosophila innate immune response have been identified. The extracellular components include a peptidoglycan recognition protein (PGRP-SA), a serine protease (Persephone), a serine protease inhibitor (Necrotic), and a putative li...

Section: Discussionmentioning

confidence: 96%

Multimerization and interaction of Toll and Spätzle in Drosophila

Yagi

Tanji

et al. 2004

112

102

“…While the hydrophobic core residues are conserved, the surface exposed residues vary greatly between TIR domains. Consequently, the distribution of surface electrostatic potential differs significantly between TIR domains (22), possibly underlying the differences in binding specificity. For TIR domains of membranous receptors, 4 structures from TLR1, TLR2, TLR10, and IL-1RAPL have been reported (8)(9)(10).…”

Section: Discussionmentioning

confidence: 99%

“…The GST-fusion proteins were purified by glutathione Sepharose 4B FF (GE Healthcare) affinity chromatography. The TIR domain of human Mal, as well as the DD of IRAK4, was purified using a modified previously reported method (22,38). These purified proteins were incubated with Glutathione Sepharose 4B (GE Healthcare) for 3 hours.…”

Section: Vector Preparationsmentioning

confidence: 99%

Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling

Ohnishi

Tochio

Kato

et al. 2009

188

185

“…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.…”

mentioning

confidence: 99%

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

Enokizono

Kumeta

Funami

et al. 2013

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...