Assembly of Oligomeric Death Domain Complexes during Toll Receptor Signaling

Abstract

The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling.The bifunctional Toll pathway in Drosophila is indispensable for embryonic development and the innate immune response to Gram-positive bacteria and fungi (1). Toll is a Type I transmembrane receptor (2) whose architecture consists of N-terminal leucine-rich repeats, a transmembrane helix, and a C-terminal TIR 2 domain. It is activated by direct binding of the ligand Spätzle (3) that is cleaved from its inactive pro-form by the serine protease Easter during development (4) and by another serine protease, Spätzle-processing enzyme, during Toll-dependent antimicrobial and fungal responses (5). Activation of the Toll pathway during development sets in motion a series of events that culminate in the translocation of the Rel/ NF-B family member Dorsal into the nucleus and the activation of the zygotic genes twist and snail on the ventral side of the embryos and the deactivation of zerknullt and decapentaplegic on the dorsal side (6), whereas activation resulting from an immune challenge leads to the nuclear translocation of the Dorsal-related immunity factor (Dif) and the subsequent transcription of many genes including those encoding the antimicrobial peptides Drosomycin and Metchnikowin (7,8). In mammals, an immune response is mounted as a result of challenge by a variety of pathogen-associated molecular patterns with the activation of a homologous Toll-like receptor pathway of which approximately 11 distinct members have been identified (9).Signaling from the activated Toll receptor to the relevant transcription factors relies on interactions between adaptor proteins that associate with the TIR domain of Toll and subsequently influence interactions between other downstream components of the pathway. In Drosophila, the immediate post-receptor events are context-dependent; during an innate immune response, for example, the proteins involved are dMyD88, Tube, and Pelle (10), whereas an additiona...

An Oligomeric Signaling Platform Formed by the Toll-like Receptor Signal Transducers MyD88 and IRAK-4

“…This arrangement is consistent with that seen in the PIDDosome in which type 2 interactions stabilize the interface between the bottom layer of PIDD subunits and the second layer of RAIDDs. The assembly process may display positive cooperativity similar to that recently demonstrated in the binding of the Drosophila IRAK-4 homologue, Pelle, with a dimer of the signaling adaptors Tube and dMyD88 (see below) (31). The assembly process may also create a scaffold for the recruitment of IRAK-1, which unlike IRAK-4 is recruited into MyD88 complexes by DD-DD interactions and does not require the ID.…”

“…In the Toll pathway, the activated receptor recruits a heterotrimer comprising the death domains of dMyD88, the homologue of hMyD88, a bifunctional DD adaptor Tube, and the kinase Pelle, which is the homologue of IRAK-4 (31,36). The DD of Pelle and dMyD88, like those of IRAK-4 and hMyD88, are monomeric.…”

Interleukin 1/Toll-like Receptor-induced Autophosphorylation Activates Interleukin 1 Receptor-associated Kinase 4 and Controls Cytokine Induction in a Cell Type-specific Manner

“…This mechanism of activation is reminiscent of that proposed for Pelle, the Drosophila analog of the IRAKs (20,27). In this model, Pelle and the adaptor protein Tube are pre-associated with the receptor Toll.…”

The Drosophila Toll Signaling Pathway

“…Upon this interaction, MyD88, an adaptor protein, Tube, and the kinase Pelle are recruited to form a MyD88-Tube-Pelle heterotrimeric complex through death domain (DD)-mediated interactions (52)(53)(54). MyD88 and Pelle do not come into contact with each other; instead, two distinct DD surfaces in the adaptor protein Tube separately bind MyD88 and Pelle (52).…”