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MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL TIR ) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88 TIR ). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88 TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MAL TIR . We demonstrate via mutagenesis that the MyD88 TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88 TIR . Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88. Using targeted mutagenesis and protein-protein crosslinking, we show that A46 interacts with MAL and MyD88 through several facets, including residues on helices α1 and α7 and the C-terminal flexible region. We propose a model in which A46 targets the MAL and MyD88 signalosome intra-strand interfaces and gradually destroys their assemblies in a concentration-dependent manner., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)