The small GTPase Arf6 is essential for the Tram/Trif pathway in TLR4 signaling.

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal(-/-) mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.

Identification of binding sites for myeloid differentiation primary response gene 88 (MyD88) and Toll-like receptor 4 in MyD88 adapter-like (Mal).

Upon activation, Toll-like receptor 4 (TLR4) binds adapter proteins, including MyD88 (myeloid differentiation primary response gene 88) and Mal (MyD88 adapter-like) for its signal transduction. TLR4 and the adapter proteins each contain a Toll/Il-1 receptor domain (TIR domain). In this study we used random mutagenesis and the mammalian two-hybrid method MAPPIT (mammalian protein-protein interaction trap) to identify mutations in Mal that disrupt its interaction with TLR4 and/or MyD88. Our study shows that four potential binding sites and the AB-loop in the Mal TIR domain all contribute to formation of the TLR4-Mal-MyD88 complex. Mutations in the symmetrical back-to-back Mal homodimer interface affect Mal homodimerization and interaction with MyD88 and TLR4. Our data suggest that Mal dimerization may lead to formation of potential binding platforms on the top and the side of the Mal dimer that bind MyD88 or TLR4. Mutations that affect the interaction of Mal with MyD88 also affect NF-κB activation induced by Mal overexpression. In MAPPIT, co-expression of the MyD88 TIR domain enhances Mal dimerization and Mal binding to TLR4. Similarly, co-expression of Mal and the MyD88 TIR domain strongly promotes dimerization of the TLR4 intracellular domain in MAPPIT. The different types of TIR-TIR interactions in the TLR4-Mal-MyD88 complex thus show cooperative binding in MAPPIT. We present plausible models for the TIR-TIR interactions in the TLR4-Mal-MyD88 complex.

Identification of interaction sites for dimerization and adapter recruitment in Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4.

Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.

Caspase-1 targets the TLR adaptor Mal at a crucial TIR-domain interaction site.

Toll-like receptors (TLRs) are crucial components of innate immunity, ensuring efficient responses against invading pathogens. After ligand binding, TLR signaling is initiated by recruitment of adaptor molecules, a step mediated by homotypic Toll-IL-1 receptor (TIR) domain interactions. Four TIR-containing TLR adaptor molecules are described, all of which are susceptible to modification and strict regulation. For example, caspase-1 is reported to cleave the TLR adaptor Mal at position D198, an event that is indispensible for Mal function. In this report, we use the mammalian two-hybrid technique MAPPIT to study the implications of Mal cleavage. We show that a Mal mutant, which mimics caspase-1 cleavage and a caspase-1-uncleavable MalD198A mutant, are abrogated in their bridging function and lose the ability to activate NF-kappaB. A MalD198E mutant is still fully functional, suggesting that caspase-1 cleavage of Mal is not necessary for Mal-mediated signaling. D198 of Mal is conserved in MyD88 and TLR4 TIR domains and the negatively charged amino acid at this position is crucial for the interactions and function of Mal, MyD88 and TLR4 TIR. Our data suggest an inhibitory, rather than an activating role for caspase-1 in Mal regulation, and show that the caspase-1 cleavage site in Mal is part of a TIR-domain interaction site.

Reconstructing the TIR Side of the Myddosome: a Paradigm for TIR-TIR Interactions.

Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the αE-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The αC'-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains.

Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G.

The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.