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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.

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.

Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.

Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.