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 (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). 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 MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

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TLRs engage numerous adaptor proteins and signaling molecules, enabling a complex series of post-translational modifications (PTMs) to mount inflammatory responses. TLRs themselves are post-translationally modified following ligand-induced activation, with this being required to relay the full spectrum of proinflammatory signaling responses. Here, we reveal indispensable roles for TLR4 Y672 and Y749 phosphorylation in mounting optimal LPS-inducible inflammatory responses in primary mouse macrophages. LPS promotes phosphorylation at both tyrosine residues, with Y749 phosphorylation being required for maintenance of total TLR4 protein levels and Y672 phosphorylation exerting its pro-inflammatory effects more selectively by initiating ERK1/2 and c-FOS phosphorylation. Our data also support a role for the TLR4-interacting membrane proteins SCIMP and the SYK kinase axis in mediating TLR4 Y672 phosphorylation to permit downstream inflammatory responses in murine macrophages. The corresponding residue in human TLR4 (Y674) is also required for optimal LPS signaling responses. Our study, thus, reveals how a single PTM on one of the most widely studied innate immune receptors orchestrates downstream inflammatory responses.

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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 ). Due to their crystal size, conventional crystallography proved to be challenging. However, through serial femtosecond crystallography (SFX) we were able to determine the structure of MyD88 crystals. Here, we present the SFX structure of the MyD88 TIR assembly, which revealed a biological relevant two-stranded higher order assembly arrangement of TIR domains analogous to that seen previously for MAL TIR . Our study provides structural and mechanistic insights into TLR signal transduction 1 .

Myeloid differentiation primary response gene 88 (MyD88) and MyD88 adaptor-like/TIRAP (MAL) are Toll-like receptor (TLR) adaptors that signal to induce proinflammatory cytokine production. The TLR responds to a pathogen or endogenous molecules identified as 'dangerous' to our cells. This results in triggering the cytoplasmic/interleukin-1 receptor followed by the recruitment of adaptor proteins MAL and MyD88. 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 Xray 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 reveals a two-stranded higherorder assembly arrangement of TIR domains analogous to that seen previously for MAL TIR . Through mutagenesis studies we reveal the MyD88 TIR assembly interfaces are critical for TLR4 signaling, in vivo, and provide evidence to show MAL promotes unidirectional assembly of MyD88 TIR . Together our results provide structural and mechanistic insight into TLR signal transduction and allows a direct comparison of the MicroED and SFX techniques on the same protein crystal [1].

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