Oral Presentation 50th Lorne Proteins Conference 2025

Structural basis for TIR domain-mediated innate immune signaling by Toll-like receptor adaptors TRIF and TRAM (115286)

Mengqi Pan 1 2 , Mohammad Manik 3 , Le Xiao 3 , Weixi Gu 1 , Hyoyoung Kim 1 , Sabrina Pospich 4 , Andrew Hedger 1 , Parimala Vajjhala 1 , Morris Lee 1 , Xiaoqi Qian 1 , Michael Landsberg 1 , Thomas Ve 5 , Jeffrey Nanson 1 6 , Stefan Raunser 4 , Katryn Stacey 1 , Hao Wu 3 , Bostjan Kobe 1
  1. School of Chemistry and Molecular Biosciences, the University of Queensland, St Lucia, QLD, Australia
  2. Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, America
  4. Max Planck Institute of Molecular Physiology, Dortmund, Germany
  5. Institute for Glycomics, Griffith University, Southport, QLD, Australia
  6. Gulbali Institute, Charles Sturt University, Wagga Wagga, NSW, Australia

 

In this study, we reveal the structural basis of MyD88-independent Toll-like receptor (TLR) signaling mediated by the TIR (Toll/interleukin-1 receptor) domain-containing adaptor proteins TRIF (TIR domain-containing adaptor-inducing interferon-β) and TRAM (TRIF-related adaptor molecule), which play essential roles in innate immunity by recognizing pathogen-associated molecular patterns. Although TRIF and TRAM are known to be crucial for TLR signaling, the structural mechanisms underlying their function have remained unclear. Here, using cryo-electron microscopy (cryo-EM) and X-ray crystallography, we provide high-resolution structural insights into filamentous assemblies formed by TRIF and TRAM TIR domains, resolved at 3.3 Å and 5.6 Å, respectively. These cryo-EM structures reveal that both TRIF and TRAM TIR domains organize into two-stranded parallel helical filaments, highlighting the role of BB-loop-mediated intrastrand and interstrand interactions critical for TLR4-mediated signaling.

To further probe the functional significance of these interactions, we used mutagenesis and functional assays, which confirmed the essential roles of these BB-loop-mediated contacts in promoting effective TLR signaling. Additionally, we report the 3Å crystal structure of a monomeric TRAM TIR domain bearing a C117H mutation in the BB loop, which reveals notable conformational differences consistent with the loss of signaling activity. This mutant structure not only underscores the functional relevance of BB-loop interactions but also provides insights into conformational changes associated with TIR domain inactivation.

Our findings suggest a unified structural mechanism for TLR signaling across the four key TIR domain-containing adaptors—MyD88, MAL, TRIF, and TRAM—by demonstrating common architectural and interaction features within these signaling assemblies. The structural and functional insights gained in this study offer potential therapeutic avenues for modulating TLR signaling in immunity-related disorders, presenting new opportunities for targeted treatment strategies.