Molecular Basis of Phosphatidylinositol 4,5-Bisphosphate Recognition by the Adaptor Protein Tirap

Abstract

Toll-like receptors (TLRs) provide a mechanism for host defense by activating innate immune responses. Activated TLRs [e.g., by bacterial lipopolysaccharide (LPS)] dimerize, and interact with adaptor proteins through their cytosolic TIR domains to trigger a signaling cascade that ultimately leads to the expression of proteins involved in pro-inflammatory responses. One such adaptor protein is the TIR domain-containing adaptor protein (TIRAP), which contains an N-terminal phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding region that is required for plasma membrane targeting and a C-terminal TIR domain, which mediates myeloid differentiation primary response gene 88 (MyD88) association. Upon ligand binding, the LPS-binding protein TLR4 is proposed to be recruited to PtdIns(4,5)P2-rich regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the plasma membrane via TIR-TIR domain interactions; thus TIRAP bridges MyD88 binding to activated TLR4. A conserved short stretch at the N-terminus of TIRAP has been shown to be sufficient to target the protein to the plasma membrane. We show that this region, which we named the PtdIns(4,5)P2 binding motif (PBM), folds in dodecylphosphocholine (DPC) micelles, and binds PtdIns(4,5)P2. The solution structure of the DPC-associated TIRAP PBM indicates that the peptide adopts a helical structure in micelles. Furthermore, we demonstrate that PtdIns(4,5)P2 can induce a helical structure in TIRAP PBM, a feature observed in other PtdIns(4,5)P2 modules. NMR data indicates that TIRAP PBM binds PIP2 in a fast exchange regime with a moderate affinity through two conserved basic regions. Thus, these studies will provide a basis for understanding the mechanism of TIRAP's membrane targeting and recruitment of TIR-containing proteins required to trigger pro-inflammatory signaling.