Constitutively oxidized CXXC motifs within the CD3 heterodimeric ectodomains of the T cell receptor complex enforce the conformation of juxtaposed segments.
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.
From the Department of Medical Oncology, Laboratory of Immunobiology, Dana-Farber Cancer Institute and Departments of Medicine and.
Kristine N
KN
Brazin
Biological Chemistry and Molecular Pharmacology, Harvard Medical School and.
Robert J
RJ
Mallis
Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115.
Chen
C
Li
From the Department of Medical Oncology, Laboratory of Immunobiology, Dana-Farber Cancer Institute and.
Derin B
DB
Keskin
Biological Chemistry and Molecular Pharmacology, Harvard Medical School and.
Haribabu
H
Arthanari
Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115.
Yuanwei
Y
Gao
Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115.
Shiaw-Lin
SL
Wu
Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115.
Barry L
BL
Karger
Biological Chemistry and Molecular Pharmacology, Harvard Medical School and.
Gerhard
G
Wagner
From the Department of Medical Oncology, Laboratory of Immunobiology, Dana-Farber Cancer Institute and Departments of Medicine and ellis_reinherz@dfci.harvard.edu.
Ellis L
EL
Reinherz
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018801:CD2 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D040542:Mechanotransduction, Cellular; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
D018801:CD2 Antigens; D017252:CD3 Complex; D004220:Disulfides; D021122:Protein Subunits; D011948:Receptors, Antigen, T-Cell
CD3; Disulfide; Mass Spectrometry (MS); Mechanotransduction; Nuclear Magnetic Resonance (NMR); Signaling; T-cell Receptor (TCR); Transmembrane