title: Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif.
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.
authors.affiliation: Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg VA, 24061, USA.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg VA, 24061, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Xiong
authors.affiliation: School of Materials and Metallurgy, Inner Mongolia University of Science and Technology, P. R. China.
authors.forename: Shuyan
authors.initials: S
authors.lastname: Xiao
authors.affiliation: Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg VA, 24061, USA.
authors.forename: Tuo-Xian
authors.initials: TX
authors.lastname: Tang
authors.affiliation: Biomolecular Magnetic Resonance Facility, University of Virginia, Charlottesville VA, 22904, USA.
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Ellena
authors.affiliation: Departments of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder CO, 80309, USA.
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Armstrong
authors.affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Tech, Blacksburg VA, 24061, USA.
authors.forename: Carla V
authors.initials: CV
authors.lastname: Finkielstein
authors.affiliation: Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg VA, 24061, USA.
authors.forename: Daniel G S
authors.initials: DG
authors.lastname: Capelluto
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D059748:Proteolysis; D017472:Receptors, Interleukin-1 / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D008562:Membrane Glycoproteins; D010716:Phosphatidylinositols; D017472:Receptors, Interleukin-1; C434984:TIRAP protein, human