MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography

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. MAL and MyD88 are downstream adaptors of Toll-like receptors (TLR) and the MAL TIR domain forms filaments in vitro, which in turn nucleate the assembly of crystalline arrays of the MyD88 TIR domain. Here, the authors present the structure of these MyD88 TIR crystalline arrays solved by both microcrystal electron diffraction and serial femtosecond crystallography, and they show with mutagenesis experiments that MyD88 interface residues are important for TLR4 signaling in vivo.