Li, Y.Pacoste, L. C.Gu, W.Thygesen, S. J.Stacey, K. J.Ve, T.Kobe, B.Xu, H.Nanson, J. D.2025-06-042025-06-041399-0047PubMed:39268708ORCID:/0000-0002-8271-3906/work/184762093http://www.scopus.com/inward/record.url?scp=85204167292&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733756562Eukaryotic TIR (Toll/interleukin-1 receptor protein) domains signal via TIR-TIR interactions, either by self-association or by interaction with other TIR domains. In mammals, TIR domains are found in Toll-like receptors (TLRs) and cytoplasmic adaptor proteins involved in pro-inflammatory signaling. Previous work revealed that the MAL TIR domain (MALTIR) nucleates the assembly of MyD88TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88TIR assembly has previously been solved, revealing a two-stranded higher-order assembly of TIR domains. In this work, it is demonstrated that the TIR domain of TLR2, which is reported to signal as a heterodimer with either TLR1 or TLR6, induces the formation of crystalline higher-order assemblies of MyD88TIRin vitro, whereas TLR1TIR and TLR6TIR do not. Using an improved data-collection protocol, the MicroED structure of TLR2TIR-induced MyD88TIR microcrystals was determined at a higher resolution (2.85Å) and with higher completeness (89%) compared with the previous structure of the MALTIR-induced MyD88TIR assembly. Both assemblies exhibit conformational differences in several areas that are important for signaling (for example the BB loop and CD loop) compared with their monomeric structures. These data suggest that TLR2TIR and MALTIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88TIR assemblies uni­directionally.This project has been funded by grants from the National Health and Medical Research Council (NHMRC; grants 2025931 to BK,1107804 to BK and TV, 1160570 to BK,KS and TV, and 1196590 to TV), the Australian Research Council (ARC;grants FL180100109 to BK and FL200100572 to TV), the Swedish Research Council (grant2019-00815 to HX) and the Knut and Alice Wallenberg Foundation(grant2018.0237 to HX).14en© 2024 International Union of Crystallography. All rights reserved.higher-order assembliesMicroEDMyD88signalosomesTIR domainsToll-like receptorsToll/interleukin-1 receptor protein202410.1107/S205979832400821085204167292