Journal:Acta Cryst D:S2059798324008210

Microcrystal electron diffraction structure of Toll-like-receptor 2 TIR domain-nucleated MyD88 TIR domain higher-order assembly

Li, Pacoste, Gu, Thygesen, Stacey, Ve, Kobe, Xu, & Nanson ^[1]

Molecular Tour
The structural insights gleaned from our study reveal a critical advancement in understanding how Toll-like receptors (TLRs) signal within the innate immune system. In this study, we concentrated on understanding how the TIR domain of TLR2 (TLR2TIR) interacts with the adaptor protein MyD88, a key player in the signaling pathways of the innate immune system. TLR2 typically signals as a heterodimer with TLR1 or TLR6 and recruits TIR-domain containing adaptors such as MAL and MyD88 to propagate inflammatory responses. Previous research demonstrated that the MAL TIR domain (MALTIR) could nucleate the assembly of MyD88^TIR into crystalline arrays in vitro, with the structure being elucidated through microcrystal electron diffraction (MicroED). Building on this, our current work reveals that TLR2^TIR, but not TLR1^TIR or TLR6^TIR, also induces the formation of these crystalline higher-order assemblies of MyD88^TIR in vitro.

By refining our data collection methods, we successfully determined the MicroED structure of TLR2 TIR-induced MyD88 TIR 8s78 microcrystals, achieving superior resolution (2.85 Å) and completeness (89%) compared to MAL TIR-induced MyD88 TIR 7beq microcrystals, comparing them. Structural alignment of TLR2^TIR- and MAL^TIR-induced MyD88^TIR assemblies (four molecules are shown). Notably, both types of assemblies showed distinct conformational differences in regions critical for signaling, such as the BB loop and CD loop, when compared to their monomeric structures. These findings suggest that TLR2^TIR and MAL^TIR interact with MyD88 in a similar manner, promoting the unidirectional nucleation of MyD88^TIR assemblies during signaling. This highlights the unique role of TLR2^TIR in modulating MyD88 assembly and signaling, offering new insights into the specificity and dynamics of TLR-mediated immune responses.

This can be seen in the significant conformational differences (e.g., BB loop, CD loop, αB helix) are observed in several regions when compared with the X-ray and NMR structures of monomeric proteins, e.g.,

Superposition of the monomer of TLR2^TIR induced MyD88^TIR structure 8s78 (blue) with the MyD88^TIR X-ray structure 4e07 yellow)

Superpostion of the monomer of TLR2^TIR induced MyD88^TIR structure 8s78 (blue) on the MyD88^TIR NMR structure 2z5v orange)

References

1. ↑ Li Y, Pacoste LC, Gu W, Thygesen SJ, Stacey KJ, Ve T, Kobe B, Xu H, Nanson JD. Microcrystal electron diffraction structure of Toll-like receptor 2 TIR-domain-nucleated MyD88 TIR-domain higher-order assembly. Acta Crystallogr D Struct Biol. 2024 Sep 1;80(Pt 9):699-712. PMID:39268708 doi:10.1107/S2059798324008210