Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif

Xiaolin Zhao,Jeffrey F Ellena,Wen Xiong,Tuo-Xian Tang,Daniel G S Capelluto,Geoffrey S Armstrong,Carla V Finkielstein,Shuyan Xiao

doi:10.1038/srep43043

Abstract

Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.

Highlights

The innate immune system is composed of germline-encoded receptor proteins that recognize invading pathogens by what is referred to as pathogen associated molecular patterns (PAMPs)[1]
We found that the TIR domain-containing adaptor protein (TIRAP) PI-binding motif (PBM) region was intrinsically disordered, but underwent a helical conformation in the presence of DPC micelles (Fig. 1B, inset)
But larger, resonance perturbations in DPC-embedded TIRAP PBM spectra were observed with the water insoluble c16-PtdIns(4,5)P2 (Supplementary Fig. S1), indicating that, during the NMR titrations, c8-PtdIns(4,5)P2 was incorporated into DPC micelles

Summary

Introduction

The innate immune system is composed of germline-encoded receptor proteins that recognize invading pathogens by what is referred to as pathogen associated molecular patterns (PAMPs)[1]. The presence of TIRAP at the plasma membrane is required even when there are low TLR ligand levels[6]. The structure of the TIRAP TIR domain reveals two potential dimerization interfaces in a configuration that allows the two monomers of the N-terminal PBD to be oriented in the same direction, facilitating PtdIns(4,5)P2-mediated plasma membrane targeting[8,9]. Plasma membrane targeting of TIRAP is likely mediated by a short stretch of basic residues [amino acids 15–35; named PI-binding motif (PBM)] within the putative PBD10. TIRAP interacts with PI3Ks, enzymes that convert PtdIns(4,5)P2 into PtdIns(3,4,5)P3, impairing TIRAP’s membrane targeting[13]. We identified basic and nonpolar residues in TIRAP PBM involved in PI interaction and demonstrated that phosphorylation in the inositol ring as well as the presence of fatty acids are required for binding. Misfolding of the membrane-interacting region of TIRAP by phosphorylation is a prerequisite for its rapid turnover

Methods

Results

Conclusion