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

Abstract

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...

Phosphatase PTPN4 Preferentially Inhibits TRIF-Dependent TLR4 Pathway by Dephosphorylating TRAM

“…Tyrosine phosphorylation then provides docking sites for other proteins (11). Structural analysis indicates that both TRIF and TRAM TIR domains form a BB-loop-mediated homodimer (30). The dimerization of TRAM TIR presents an interaction surface for TRIF (30).…”

“…Structural analysis indicates that both TRIF and TRAM TIR domains form a BB-loop-mediated homodimer (30). The dimerization of TRAM TIR presents an interaction surface for TRIF (30). Tyrosine phosphorylation within the TIR domain of TRAM may initiate the conformational change and then provide docking sites to facilitate the interaction between TRAM and TRIF.…”

“…5D). The structure of TIR domain of TRAM from human has been determined (30). We analyzed this structure and noted that both Y-154 and Y-167 were surface exposed (Fig.…”

Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type–Specific Manner

“…TIRAP-dependent binding of MyD88 recruits the signaling molecules IL-1R-associated kinases 1 and 4, which forms so-called myddosomes that activate NF-kB and MAPK signaling cascades, leading to production of proinflammatory cytokines (6,7). While TIRAP-MyD88-dependent signaling occurs at the plasma membrane, Toll-IL-1R domaincontaining adaptor molecule-2 (TICAM-2; also called TRAM)--and TICAM-1 (also called TRIF)-dependent IFN-regulatory factor 3 (IRF3) activation initiates from the endosomes (8)(9)(10)(11)(12)(13)(14). Dissociation from TIRAP-MyD88 and subsequent clathrin-and dynamin-mediated internalization of LPS-TLR4 are prerequisites for binding to the sorting adaptor TICAM-2 that localizes to the endosomal membrane via myristoylation at the N terminus (12,15).…”

Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2

“…According to the structural data, the E87/D88/D89 motif located in the loop region between the bA sheet and the aA helix and S156 in the aC helix region contributes to TICAM-1-TICAM-2 heterodimerization (Supplemental Fig. 1) (33). In multiple alignments of TICAM-2 sequences, these motifs and two other acidic residues, D91 and E92, were highly conserved among various mammalian species (Fig.…”