Structural Basis of Phosphoinositide (PIP) Recognition by the TIRAP PIP-Binding Motif

Abstract

Toll-like receptors (TLRs) provide early immune system recognition and response to infection. TLRs activated by pathogens consequentially initiate a cytoplasmic signaling cascade though adaptor proteins, the first being the modular TIR domain-containing adaptor protein (TIRAP). TIRAP contains a C-terminal TIR domain, which is responsible for association with TLRs and other adaptors including the myeloid differentiation primary response gene88 (MyD88) protein. Membrane recruitment of TIRAP is mediated by its N-terminal PIP-binding motif (PBM). Upon ligand-mediated activation, TLRs are recruited to the PIP-enriched regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the membrane by bridging MyD88 to activate the TLR signaling pathway. To understand the mechanism of membrane targeting of TIRAP and the basis for its regulation, we functionally and structurally characterized its PBM using experimental and computational studies. TIRAP PBM adopts a folded conformation in membrane mimics, such as dodecylphosphocholine micelles, and binds PIPs. Structural rearrangements of TIRAP PBM were influenced by membrane interaction, with monodispersed PIPs inducing helical structure in the peptide. In contrast, monodispersed phosphatidylinositol and inositol trisphosphate did not promote structural changes in TIRAP PBM. NMR spectra reveal that TIRAP PBM binds PIPs in a fast exchange regime with a moderate affinity through two conserved basic regions. Solution NMR structure of TIRAP PBM shows a central short helix, and paramagnetic studies indicate that this region is close to the micelle core. Molecular dynamics simulations studies indicated that TIRAP PBM diffused to and interacted with a model membrane composed of palmitoyl oleoyl phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. Thus, we propose that two sets of basic residues contact both the head group and acyl chains of PIPs, whereas the central helix is responsible for membrane insertion.