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

Clabbers, Max T. B.; Holmes, Susannah; Muusse, Timothy W; Vajjhala, Parimala R.; Thygesen, Sara J.; Malde, Alpeshkumar K.; Hunter, Dominic J. B.; Croll, Tristan I.; Flueckiger, Leonie; Nanson, J.D.; Rahaman, Md. Habibur; Aquila, Andrew; Hunter, Mark S.; Liang, Mao; Yoon, Chun Hong; Zhao, Jingjing; Zatsepin, Nadia A.; Abbey, Brian; Sierecki, Emma; Gambin, Yann; Stacey, Katryn J.; Darmanin, Connie; Kobe, Boštjan; Xu, Hongyi; Ve, Thomas

doi:10.1038/s41467-021-22590-6

Cited by 63 publications

(56 citation statements)

References 138 publications

(111 reference statements)

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“…The hierarchy of myddosome assembly is a matter of controversy. Recent evidence obtained by microcrystal electron diffraction and serial femtosecond crystallography favors a sequential model in which TIRAP provides a platform for the unidirectional assembly of MyD88 oligomers [ 68 ], whereas the observation of pre-myddosome scaffolds based on DD interactions have led to the hypothesis that MyD88 is pre-assembled before TLR4 activation [ 69 ]. Indeed, ERMES-associated MyD88 clusters observed here in the yeast cell seem to form even when key TIR residues are mutated, so they might be composed of DD-driven ordered structures.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae

et al. 2021

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Toll-like receptor (TLR) signaling is key to detect pathogens and initiating inflammation. Ligand recognition triggers the assembly of supramolecular organizing centers (SMOCs) consisting of large complexes composed of multiple subunits. Building such signaling hubs relies on Toll Interleukin-1 Receptor (TIR) and Death Domain (DD) protein-protein interaction domains. We have expressed TIR domain-containing components of the human myddosome (TIRAP and MyD88) and triffosome (TRAM and TRIF) SMOCs in Saccharomyces cerevisiae, as a platform for their study. Interactions between the TLR4 TIR domain, TIRAP, and MyD88 were recapitulated in yeast. Human TIRAP decorated the yeast plasma membrane (PM), except for the bud neck, whereas MyD88 was found at cytoplasmic spots, which were consistent with endoplasmic reticulum (ER)-mitochondria junctions, as evidenced by co-localization with Mmm1 and Mdm34, components of the ER and Mitochondria Encounter Structures (ERMES). The formation of MyD88-TIRAP foci at the yeast PM was reinforced by co-expression of a membrane-bound TLR4 TIR domain. Mutations in essential residues of their TIR domains aborted MyD88 recruitment by TIRAP, but their respective subcellular localizations were unaltered. TRAM and TRIF, however, did not co-localize in yeast. TRAM assembled long PM-bound filaments that were disrupted by co-expression of the TLR4 TIR domain. Our results evidence that the yeast model can be exploited to study the interactions and subcellular localization of human SMOC components in vivo.

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Section: Discussionmentioning

confidence: 99%

Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae

et al. 2021

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“…The group includes TIR domains from mammalian membrane receptors (TLRs and IL-1Rs), as well as those from the cytoplasmic adaptor proteins. A number of crystal structures of TIR domains from this group have been determined, but the structural basis of their self-assembly only became clear through the structural studies of higher-order structures reconstituted for the adaptors MAL and MyD88 ( 26 , 27 ).…”

Section: Scaffold Tir-domain Assembliesmentioning

confidence: 99%

“…Reconstitution of higher-order assemblies of MAL and MyD88 TIR domains yielded filamentous and micro-crystalline complexes, respectively ( 26 , 27 ). These reconstitution experiments correlated with the functional signaling pathway, as the TLR4 TIR domain seeded the assembly of MAL TIR domains, while MAL TIR domains seeded the assembly of MyD88 TIR domains.…”

Section: Scaffold Tir-domain Assembliesmentioning

confidence: 99%

“…These reconstitution experiments correlated with the functional signaling pathway, as the TLR4 TIR domain seeded the assembly of MAL TIR domains, while MAL TIR domains seeded the assembly of MyD88 TIR domains. Structure determination of these higher-order assemblies, combined with mutagenesis and signaling assays, has provided clarity on the biologically relevant association of scaffold TIR domains ( 26 , 27 ). These structures feature two parallel strands of TIR domains, each showcasing a head-to-tail arrangement of TIR domains, held together through a BB-loop-mediated “BE” intrastrand interface.…”

Section: Scaffold Tir-domain Assembliesmentioning

confidence: 99%

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Structural Evolution of TIR-Domain Signalosomes

Nimma

Maruta

et al. 2021

Front. Immunol.

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TIR (Toll/interleukin-1 receptor/resistance protein) domains are cytoplasmic domains widely found in animals and plants, where they are essential components of the innate immune system. A key feature of TIR-domain function in signaling is weak and transient self-association and association with other TIR domains. An additional new role of TIR domains as catalytic enzymes has been established with the recent discovery of NAD+-nucleosidase activity by several TIR domains, mostly involved in cell-death pathways. Although self-association of TIR domains is necessary in both cases, the functional specificity of TIR domains is related in part to the nature of the TIR : TIR interactions in the respective signalosomes. Here, we review the well-studied TIR domain-containing proteins involved in eukaryotic immunity, focusing on the structures, interactions and their corresponding functional roles. Structurally, the signalosomes fall into two separate groups, the scaffold and enzyme TIR-domain assemblies, both of which feature open-ended complexes with two strands of TIR domains, but differ in the orientation of the two strands. We compare and contrast how TIR domains assemble and signal through distinct scaffolding and enzymatic roles, ultimately leading to distinct cellular innate-immunity and cell-death outcomes.

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“…To alleviate radiation damage, researchers showed that the structure model can be improved by merging small-wedge 3D ED datasets collected from multiple crystals [28]. Furthermore, recent results showed that it is even possible to solve unknown protein structures (phased from a homologue less than 40% in sequence identity or having different conformation compared to the molecular replacement model) by MicroED [29,30], and visualize ligand binding interactions with electron diffraction datasets [31].…”

Section: Introductionmentioning

confidence: 99%

A Comparison of Structure Determination of Small Organic Molecules by 3D Electron Diffraction at Cryogenic and Room Temperature

et al. 2021

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3D electron diffraction (3D ED), also known as micro-crystal electron diffraction (MicroED), is a rapid, accurate, and robust method for structure determination of submicron-sized crystals. 3D ED has mainly been applied in material science until 2013, when MicroED was developed for studying macromolecular crystals. MicroED was considered as a cryo-electron microscopy method, as MicroED data collection is usually carried out in cryogenic conditions. As a result, some researchers may consider that 3D ED/MicroED data collection on crystals of small organic molecules can only be performed in cryogenic conditions. In this work, we determined the structure for sucrose and azobenzene tetracarboxylic acid (H4ABTC). The structure of H4ABTC is the first crystal structure ever reported for this molecule. We compared data quality and structure accuracy among datasets collected under cryogenic conditions and room temperature. With the improvement in data quality by data merging, it is possible to reveal hydrogen atom positions in small organic molecule structures under both temperature conditions. The experimental results showed that, if the sample is stable in the vacuum environment of a transmission electron microscope (TEM), the data quality of datasets collected under room temperature is at least as good as data collected under cryogenic conditions according to various indicators (resolution, I/σ(I), CC1/2 (%), R1, Rint, ADRA).

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

Cited by 63 publications

References 138 publications

Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae

Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae

Structural Evolution of TIR-Domain Signalosomes

A Comparison of Structure Determination of Small Organic Molecules by 3D Electron Diffraction at Cryogenic and Room Temperature

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