Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling

Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):E6480-E6489. doi: 10.1073/pnas.1701868114. Epub 2017 Jul 24.


MyD88 adaptor-like (MAL) is a critical protein in innate immunity, involved in signaling by several Toll-like receptors (TLRs), key pattern recognition receptors (PRRs). Crystal structures of MAL revealed a nontypical Toll/interleukin-1 receptor (TIR)-domain fold stabilized by two disulfide bridges. We therefore undertook a structural and functional analysis of the role of reactive cysteine residues in the protein. Under reducing conditions, the cysteines do not form disulfides, but under oxidizing conditions they are highly amenable to modification. The solution structure of the reduced form of the MAL TIR domain, determined by NMR spectroscopy, reveals a remarkable structural rearrangement compared with the disulfide-bonded structure, which includes the relocation of a β-strand and repositioning of the functionally important "BB-loop" region to a location more typical for TIR domains. Redox measurements by NMR further reveal that C91 has the highest redox potential of all cysteines in MAL. Indeed, mass spectrometry revealed that C91 undergoes glutathionylation in macrophages activated with the TLR4 ligand lipopolysaccharide (LPS). The C91A mutation limits MAL glutathionylation and acts as a dominant negative, blocking the interaction of MAL with its downstream target MyD88. The H92P mutation mimics the dominant-negative effects of the C91A mutation, presumably by preventing C91 glutathionylation. The MAL C91A and H92P mutants also display diminished degradation and interaction with interleukin-1 receptor-associated kinase 4 (IRAK4). We conclude that in the cell, MAL is not disulfide-bonded and requires glutathionylation of C91 for signaling.

Keywords: MAL/TIRAP; NMR spectrometry; Toll-like receptor; glutathione; inflammation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism
  • Glutathione / chemistry
  • Glutathione / genetics
  • Glutathione / metabolism*
  • HEK293 Cells
  • Humans
  • Membrane Glycoproteins* / chemistry
  • Membrane Glycoproteins* / genetics
  • Membrane Glycoproteins* / metabolism
  • Mutation, Missense
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Domains
  • Protein Processing, Post-Translational*
  • Protein Structure, Secondary
  • Receptors, Interleukin-1* / chemistry
  • Receptors, Interleukin-1* / genetics
  • Receptors, Interleukin-1* / metabolism
  • Signal Transduction*
  • Structure-Activity Relationship


  • Membrane Glycoproteins
  • Receptors, Interleukin-1
  • TIRAP protein, human
  • Glutathione
  • Cysteine

Associated data

  • PDB/2Y92
  • PDB/2NDH