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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

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

Xiaolin Zhao1, Wen Xiong1, Shuyan Xiao2

  • 1Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg VA, 24061, USA.

Scientific Reports
|February 23, 2017
PubMed

Insights

Toll-like receptors (TLRs) signal innate immunity. This study reveals how TIRAP protein binds membranes via its PBM, and how phosphorylation regulates this binding, controlling immune response duration.

Area of Science:

  • Immunology
  • Cell Biology
  • Structural Biology

Background:

  • Pathogen-activated Toll-like receptors (TLRs) initiate innate immune responses by recruiting adaptor proteins like TIRAP to the plasma membrane.
  • TIRAP's membrane recruitment is crucial for downstream signaling but requires precise regulation to prevent excessive immune activation.

Purpose of the Study:

  • To elucidate the structural basis of TIRAP's membrane recruitment mediated by its phosphoinositide-binding motif (PBM).
  • To investigate the regulatory mechanism controlling TIRAP's interaction with the cell membrane and its subsequent removal.

Main Methods:

  • Investigated conformational changes of the TIRAP PBM using zwitterionic micelles and purified phosphoinositides (PIs).
  • Analyzed PI-binding interactions through residue-specific binding studies.
  • Examined the effect of phosphorylation at Thr28 on PBM structure and PI binding affinity.

Main Results:

  • The TIRAP PBM transitions from a disordered to a helical structure upon binding to PIs, mediated by specific basic and nonpolar residues.
  • Phosphorylation of TIRAP at Thr28 within the PBM induces a conformational change, distorting the helix and reducing PI binding.
  • This phosphorylation-induced structural change impairs membrane targeting and promotes TIRAP degradation, limiting innate immune signaling.

Conclusions:

  • The study provides a structural mechanism for TIRAP membrane insertion via PI binding.
  • Phosphorylation acts as a key regulatory switch, controlling TIRAP's membrane association and duration of innate immune responses.

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