Structural Basis of Membrane Targeting by the Innate Immunity Adaptor TIRAP

Abstract

Toll-like receptors (TLRs) are responsible for early immune system recognition and response to infection. Pathogen-activated TLRs trigger a cytoplasmic signaling cascade though adaptor proteins, starting with the 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 phosphoinositide (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 to activate the TLR signaling pathway. To understand the mechanism of TIRAP membrane targeting and the basis for its regulation, we functionally and structurally characterized TIRAP and its PBM using biophysical approaches. We show that TIRAP's PBM adopts a helical conformation in the presence of dodecylphosphocholine (DPC) micelles. NMR studies revealed that TIRAP PBM binds PIPs through its two conserved basic termini following a fast-exchange regime with moderate affinity. Mutation of these two basic regions abolishes PIP binding without distorting the helical structure of the peptide. Interestingly, association of TIRAP PBM to PIPs requires the acyl chains of the lipid. The solution NMR structure of TIRAP PBM exhibits a central relatively hydrophobic helix surrounded by the flexible N- and C-termini. Paramagnetic studies indicate that the helix is close to the micelle core, whereas the N- and C- termini are located on the micellar surface. Nuclear spin relaxation experiments indicate that this N- and C-termini of TIRAP PBM become more ordered when bound to PIPs. Thus, we propose that the central helix in PBM is responsible for membrane insertion, whereas the two sets of basic residues interact with PIPs to stabilize TIRAP's membrane interaction.