myd88 tir domain higher order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography

• DOI: 10.17863/cam.69652
• Publication Date: 2021-05-10
• Authors: Timothy W. Muusse; Alpeshkumar K. Malde; Nadia A. Zatsepin; Jeffrey D. Nanson; Brian Abbey; Leonie Flueckiger; Dominic J. B. Hunter; Dominic J. B. Hunter; Katryn J. Stacey; Tristan I. Croll; Max T. B. Clabbers; Max T. B. Clabbers; Emma Sierecki; Hongyi Xu; Parimala R. Vajjhala; Thomas Ve; Andrew Aquila; Connie Darmanin; Mengning Liang; Mark S. Hunter; Md. Habibur Rahaman; Bostjan Kobe; Jingjing Zhao; Susannah Holmes; Sara J. Thygesen; Yann Gambin; Chun Hong Yoon
• Keywords: Models, Molecular; Protein Conformation, alpha-Helical; 0301 basic medicine; 570; 572; /631/45/535; /145; Science; /631/535/1258/1259; 610; Molecular Dynamics Simulation; /96/31; Article; /96/95; /82/80; 03 medical and health sciences; Engineering; Protein Domains; Humans; /82/83; /631/250/262/2106/2108; 0303 health sciences; Crystallography; Membrane Glycoproteins; Q; /101/28; article; 500; Receptors, Interleukin-1; Recombinant Proteins; Physical sciences; Toll-Like Receptor 4; HEK293 Cells; Mutation; Myeloid Differentiation Factor 88; Protein Conformation, beta-Strand; /631/535/1266/1265; Dimerization; info:eu-repo/classification/ddc/500; Signal Transduction

AbstractMyD88 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.