Garcia K C, Tallquist M D, Pease L R, Brunmark A, Scott C A, Degano M, Stura E A, Peterson P A, Wilson I A, Teyton L
Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13838-43. doi: 10.1073/pnas.94.25.13838.
Cellular immunity is mediated by the interaction of an alphabeta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alphabeta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alphabeta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alphabeta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10(-4) to 10(-6)M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.
细胞免疫由αβ T细胞受体(TCR)与在主要组织相容性复合体(MHC)分子背景下呈递的肽相互作用介导。同种异体反应性T细胞具有αβ TCR,其可识别自身和外来的肽 - MHC(pMHC)复合体,这意味着TCR与不同的pMHC具有显著的互补性。为了表征同种异体反应性TCR识别pMHC的分子基础,我们在果蝇细胞中制备了一种可溶性重组形式的同种异体反应性αβ T细胞受体。这种重组TCR,即2C,以正确配对的αβ异二聚体形式表达,其链通过C末端区域的二硫键共价连接。通过使用构象特异性单克隆抗体以及同基因和异基因pMHC配体,通过表面等离子体共振(SPR)分析探究了2C TCR的天然构象。2C与H-2Kb-dEV8、H-2Kbm3-dEV8、H-2Kb-SIYR和H-2Ld-p2Ca的相互作用对于同基因(H-2Kb)和异基因(H-2Kbm3、H-2Ld)配体的亲和力范围为Kd = 10^(-4)至10^(-6)M。一般来说,同基因配体的结合亲和力比异基因配体弱,这与当前胸腺选择和T细胞活化的阈值模型一致。2C TCR的结晶需要对α链和β链的C末端残基进行蛋白水解修剪。已生长出2C与H-2Kb-dEV8、H-2Kbm3-dEV8和H-2Kb-SIYR复合物的X射线质量晶体。