Abstract
Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu(183), Ser(244), and Arg(288) impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88151-189 and GFP-MyD88168-189), comprising the Glu(183) residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.
Highlights
myeloid differentiation factor 88 (MyD88) is an adaptor protein that plays a crucial role in the immune response
We focused on residues present within the flanking regions of three highly conserved motifs denoted box 1, 2, and 3 [17] of the MyD88-Toll/IL-1 receptor (TIR), with specific attention to charged/polar residues that are likely to be involved in protein-protein interactions (Fig. 1A)
We selected other residues within the AB and CD loops, as they are located in exposed regions that could play a role in the TIR-TIR interface of the MyD88 dimer
Summary
MyD88 is an adaptor protein that plays a crucial role in the immune response. Results: We identified residues within the TIR domain of MyD88 required for protein self-association. Our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells. TLRs are type I transmembrane proteins and comprise an extracellular domain, which mediates the recognition of pathogen-associated molecular patterns, a transmembrane region, and an intracellular Toll-IL-1 receptor (TIR) domain that activates downstream signaling pathways [5]. TIR domains are crucial for TLR/IL-1R signal transduction as they mediate receptor-receptor, receptor-adaptor, and adaptor-adaptor interactions For this reason, they have been the subject of numerous structural and functional studies aimed at identifying the residues participating to homo and hetero interactions [21,22,23,24,25,26]. Our findings identified a novel region of the TIR domain of MyD88 that is crucial for TLR/IL-1R signal transduction
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