Constitutively oxidized CXXC motifs within the CD3 heterodimeric ectodomains of the T cell receptor complex enforce the conformation of juxtaposed segments

J Biol Chem. 2014 Jul 4;289(27):18880-92. doi: 10.1074/jbc.M114.574996. Epub 2014 May 21.


The CD3ϵγ and CD3ϵδ heterodimers along with the CD3ζζ homodimer are the signaling components of the T cell receptor (TCR). These invariant dimers are non-covalently associated on the T cell plasma membrane with a clone-specific (i.e. clonotypic) αβ heterodimer that binds its cognate ligand, a complex between a particular antigenic peptide, and an MHC molecule (pMHC). These four TCR dimers exist in a 1:1:1:1 stoichiometry. At the junction between the extracellular and transmembrane domains of each mammalian CD3ϵ, CD3γ, and CD3δ subunit is a highly conserved CXXC motif previously found to be important for thymocyte and T cell activation. The redox state of each CXXC motif is presently unknown. Here we show using LC-MS and a biotin switch assay that these CXXC segments are constitutively oxidized on resting and activated T cells, consistent with their measured reduction potential. NMR chemical shift perturbation experiments comparing a native oxidized CD3δ CXXC-containing segment with that of a mutant SXXS-containing CD3δ segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)) micelles show extensive chemical shift differences in residues within the membrane-proximal motif as well as throughout the transmembrane and cytoplasmic domains as a result of the elimination of the native disulfide. Likewise, direct comparison of the native CD3δ segment in oxidizing and reducing conditions reveals numerous spectral differences. The oxidized CXXC maintains the structure within the membrane-proximal stalk region as well as that of its contiguous transmembrane and cytoplasmic domain, inclusive of the ITAM (immunoreceptor tyrosine-based activation motif) involved in signaling. These results suggest that preservation of the CD3 CXXC oxidized state may be essential for TCR mechanotransduction.

Keywords: CD3; Disulfide; Mass Spectrometry (MS); Mechanotransduction; Nuclear Magnetic Resonance (NMR); Signaling; T-cell Receptor (TCR); Transmembrane.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • CD2 Antigens / metabolism
  • CD3 Complex / chemistry*
  • CD3 Complex / metabolism*
  • Cell Membrane / metabolism
  • Cytoplasm / metabolism
  • Disulfides / chemistry
  • Humans
  • Jurkat Cells
  • Lymphocyte Activation
  • Mechanotransduction, Cellular
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Protein Multimerization*
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Receptors, Antigen, T-Cell / metabolism*
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism


  • CD2 Antigens
  • CD3 Complex
  • Disulfides
  • Protein Subunits
  • Receptors, Antigen, T-Cell

Associated data

  • PDB/1XMW