Formation and biochemical characterization of tube/pelle death domain complexes: Critical regulators of postreceptor signaling by the Drosophila Tollreceptor

In Drosophila, the Toll receptor signaling pathway is required for embryoni c dorso-ventral patterning and at later developmental stages for innate imm une responses. It is thought that dimerization of the receptor by binding o f the ligand spatzle causes the formation of a postreceptor activation comp lex at the cytoplasmic surface of the membrane. Two components of this comp lex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thoug ht that pelle is bound by tube during formation of the activation complexes , and that this interaction is mediated by the death domains. In this paper , we show using the yeast two-hybrid system that the wild-type tube and pel le death domains bind together. Mutant tube proteins which do not support s ignaling in the embryo are also unable to bind pelle in the 2-hybrid assay, We have purified proteins corresponding to the death domains of tube and p elle and show that these form corresponding heterodimeric complexes in vitr o. Partial proteolysis reveals a smaller core consisting of the minimal dea th domain sequences. We have studied the tube/pelle interaction with the te chniques of surface plasmon resonance, analytical ultracentrifugation and i sothermal titration calorimetry. These measurements produce a value of K-d for the complex of about 0.5 mu M.