Constitutively Oxidized CXXC Motifs within the CD3 Heterodimeric Ectodomains of the T Cell Receptor Complex Enforce the Conformation of Juxtaposed Segments
Constitutively Oxidized CXXC Motifs within the CD3 Heterodimeric Ectodomains of the T Cell Receptor Complex Enforce the Conformation of Juxtaposed Segments
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DOI：
10.1074/jbc.m114.574996
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发表时间：
2014-07-11
影响因子：
4.8
通讯作者：
Reinherz, Ellis L.
中科院分区：
文献类型：
--
作者：
Brazin, Kristine N.;Mallis, Robert J.;Reinherz, Ellis L.
CD 3 β γ和CD 3 β δ异二聚体沿着CD 3 zeta zeta同二聚体是T细胞受体（TCR）的信号传导组分。这些不变二聚体在T细胞质膜上与结合其同源配体的克隆特异性（即克隆型）α β异二聚体非共价缔合，所述同源配体是特定抗原肽与MHC分子（pMHC）之间的复合物。这四种TCR二聚体以1：1：1：1化学计量存在。在每一个CD 3 γ、CD 3 γ和CD 3 δ亚基的胞外和跨膜结构域之间的连接处是高度保守的CXXC基序，先前发现其对胸腺细胞和T细胞活化是重要的。每个CXXC基序的氧化还原状态目前是未知的。在这里，我们使用LC-MS和生物素开关试验表明，这些CXXC片段在静息和活化的T细胞上被组成性氧化，与其测量的还原电位一致。NMR化学位移扰动实验比较LPPG中天然氧化的含CD 3 δ CXXC的区段与突变的含SXXS的CD 3 δ区段（1-棕榈酰-sn-甘油-3-磷酸-（1 '-消旋甘油）（钠盐））胶束在膜内的残留物中显示出广泛的化学位移差异-由于天然二硫键的消除，近端基序以及整个跨膜和细胞质结构域。同样，在氧化和还原条件下直接比较天然CD 3 δ片段揭示了许多光谱差异。氧化的CXXC保持膜近端茎区内的结构以及其连续的跨膜和胞质结构域的结构，包括参与信号传导的ITAM（基于免疫受体酪氨酸的激活基序）。这些结果表明，CD 3 CXXC氧化态的保存可能是TCR机械转导所必需的。
The CD3 epsilon gamma and CD3 epsilon delta heterodimers along with the CD3 zeta zeta 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) alpha beta 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 mammalianCD3 epsilon, CD3 gamma, and CD3 delta 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 CD3 delta CXXC-containing segment with that of a mutant SXXS-containing CD3 delta segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1'-racglycerol) (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 CD3 delta 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.