Structural basis for TIR domain-mediated innate immune signaling by Toll-like receptor adaptors TRIF and TRAM

Abstract

Innate immunity relies on Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns. The TIR (Toll/interleukin-1 receptor) domain-containing TLR adaptors TRIF (TIR domain-containing adaptor-inducing interferon-β) and TRAM (TRIF-related adaptor molecule) are essential for MyD88-independent TLR signaling. However, the structural basis of TRIF and TRAM TIR domain-based signaling remains unclear. Here, we present cryo-EM structures of filaments formed by TRIF and TRAM TIR domains at resolutions of 3.3 Å and 5.6 Å, respectively. Both structures reveal two-stranded parallel helical arrangements. Functional studies underscore the importance of intrastrand interactions, mediated by the BB-loop, and interstrand interactions in TLR4-mediated signaling. We also report the crystal structure of the monomeric TRAM TIR domain bearing the BB loop mutation C117H, which reveals conformational differences consistent with its inactivity. Our findings suggest a unified signaling mechanism by the TIR domains of the four signaling TLR adaptors MyD88, MAL, TRIF, and TRAM and reveal potential therapeutic targets for immunity-related disorders.

Keywords: MyD88-independent TLR signaling; TIR domain; cryo-EM; helical reconstruction.

Fig. 1.

TRIF and TRAM self-assemble into filaments in vitro. (A) Domain architecture of TRIF and TRAM. (B) Negative-stain EM analysis for the TRIF^∆RHIM-GFP, TRIF^∆RHIM, TRIF^TIR, TRAM^TIR, and TRAM^FL filaments. Blue arrows indicate filaments. (C) Cryo-EM reconstructions of the TRIF^∆RHIM and TRAM^TIR filaments, superimposed with the final atomic models, respectively. The N and C termini are highlighted with green and red circle/triangles, respectively.

Fig. 2.

Cryo-EM structures of TRIF^TIR-Fil and TRAM^TIR filaments. (A) Two-stranded parallel head-to-tail TRIF^TIR-Fil filamentous assembly is stabilized by the intrastrand BE and the interstrand BCD interfaces. Internal salt bridges are presented as orange dashed lines. (B) Two-stranded parallel head-to-tail TRAM^TIR filamentous assembly is stabilized by the intrastrand BE and the interstrand BCD interfaces. Internal salt bridges are presented as orange dashed lines.

Fig. 3.

Structural superposition of individual subunits of TRIF^TIR-Fil (blue) and TRAM^TIR (pink) from the respective filaments onto the two-stranded parallel filamentous assemblies (Top row) of MAL (cyan, PDB: 5UZB), MyD88 (gray, PDB: 7BEQ), AbTir (orange, PDB: 7UXU), and SPARTA (purple, PDB: 8IT1) and two-stranded antiparallel filamentous assemblies (Bottom row) of SARM1 (green, PDB: 7NAK), ROQ1 (red, PDB: 7JLX), and RPP1 (yellow, PDB: 7DFV).

Fig. 4.

Analysis of effects of mutations of residues in the TRAM:TRAM interaction interfaces on signaling efficiency. (A) MyD88 KO reporter cells were transiently transfected with 100 ng TRAM-GFP constructs (WT or mutants). On the following day, the cells were treated with or without 100 ng/ml LPS and incubated for 16 h. The cells were harvested and run on a flow cytometer. Cells expressing TRAM-GFP at a modest level were selected for analysis (SI Appendix, Fig. S5A). NF-κB-driven mScarlet-I reporter expression for each mutant was normalized to LPS-stimulated WT control (% WT + LPS). Results are from 2 to 3 independent experiments. Each dot indicates the mean of duplicates within an individual experiment. Note that WT controls were done 9 times, as they were included for every single experiment. ****P < 0.0001, ***P < 0.001, **P < 0.01, or *P < 0.05 compared to WT + LPS in one sample t test. (B) All the mutated residues are mapped on the TRAM^TIR structure and highlighted according to their effect on the signaling. The blue lines represent the physical contact area between TRAMs (identified by using the PyMOL software); for BCD interfaces, the interactions with two molecules are shown by two different shades of blue. Note that the effects of Q190A and M158R were chosen for those residues as more than one mutant was tested.

Fig. 5.

Schematic diagrams of the mechanisms of signaling by (A) TLR4 and the adaptors TRAM and TRIF and (B) TLR3 and the adaptor TRIF. Only the TIR domains of the adaptors are shown (TLR4^TIR: purple (AlphaFold two-predicted model); TLR3^TIR: blue (AlphaFold 2-predicted model); TRIF^TIR-Fil: gray; TRAM^TIR: orange). Dimerized TLR TIR domains serve as templates to recruit the adaptor proteins through structurally analogous filamentous assemblies and initiate downstream signaling. TRAF3/6: tumor necrosis factor receptor-associated factor 3/6; IFN: interferon.