Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization.
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.
Garcia
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KC
Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Tallquist
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MD
Pease
L R
LR
Brunmark
A
A
Scott
C A
CA
Degano
M
M
Stura
E A
EA
Peterson
P A
PA
Wilson
I A
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Teyton
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D000595:Amino Acid Sequence; D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D004331:Drosophila melanogaster / Q000235:genetics / Q000276:immunology; D006183:H-2 Antigens / Q000378:metabolism; D066298:In Vitro Techniques; D007519:Isoantigens; D008024:Ligands; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D006183:H-2 Antigens; D007519:Isoantigens; D008024:Ligands; D009842:Oligopeptides; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins