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

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

doi:10.1038/s41467-021-22590-6

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

Nat Commun. 2021 May 10;12(1):2578. doi: 10.1038/s41467-021-22590-6.

Authors

Max T B Clabbers^#^{1

2}, Susannah Holmes^#³, Timothy W Muusse⁴, Parimala R Vajjhala⁴, Sara J Thygesen⁴, Alpeshkumar K Malde⁵, Dominic J B Hunter^{4

6

7}, Tristan I Croll⁸, Leonie Flueckiger³, Jeffrey D Nanson⁴, Md Habibur Rahaman⁴, Andrew Aquila⁹, Mark S Hunter⁹, Mengning Liang⁹, Chun Hong Yoon⁹, Jingjing Zhao¹, Nadia A Zatsepin³, Brian Abbey³, Emma Sierecki⁶, Yann Gambin⁶, Katryn J Stacey^{4

7

10}, Connie Darmanin¹¹, Bostjan Kobe^{12

13

14}, Hongyi Xu¹⁵, Thomas Ve¹⁶

Affiliations

¹ Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden.
² Department of Biological Chemistry, University of California Los Angeles, Los Angeles, California, USA.
³ Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
⁴ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.
⁵ Institute for Glycomics, Griffith University, Southport, Queensland, Australia.
⁶ EMBL Australia Node in Single Molecule Science, University of New South Wales, Kensington, New South Wales, Australia.
⁷ Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
⁸ Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
⁹ Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, USA.
¹⁰ Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia.
¹¹ Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia. c.darmanin@latrobe.edu.au.
¹² School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia. b.kobe@uq.edu.au.
¹³ Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. b.kobe@uq.edu.au.
¹⁴ Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland, Australia. b.kobe@uq.edu.au.
¹⁵ Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden. hongyi.xu@mmk.su.se.
¹⁶ Institute for Glycomics, Griffith University, Southport, Queensland, Australia. t.ve@griffith.edu.au.

^# Contributed equally.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Crystallography / methods*
Dimerization
HEK293 Cells
Humans
Membrane Glycoproteins / chemistry*
Membrane Glycoproteins / genetics
Models, Molecular
Molecular Dynamics Simulation
Mutation
Myeloid Differentiation Factor 88 / chemistry*
Myeloid Differentiation Factor 88 / genetics
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Domains
Receptors, Interleukin-1 / chemistry*
Receptors, Interleukin-1 / genetics
Recombinant Proteins
Signal Transduction / genetics
Toll-Like Receptor 4 / chemistry*
Toll-Like Receptor 4 / genetics

Substances

MYD88 protein, human
Membrane Glycoproteins
Myeloid Differentiation Factor 88
Receptors, Interleukin-1
Recombinant Proteins
TIRAP protein, human
TLR4 protein, human
Toll-Like Receptor 4

Abstract

Publication types

MeSH terms

Substances

Grants and funding