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. 1997 Dec 9;94(25):13838-43.
doi: 10.1073/pnas.94.25.13838.

Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization

K C Garcia  1 , M D Tallquist, L R Pease, A Brunmark, C A Scott, M Degano, E A Stura, P A Peterson, I A Wilson, L Teyton
Affiliations

Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization

K C Garcia et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Expression of the 2C TCR as a covalently linked αβ heterodimer from Drosophila melanogaster cells. (A) Schematic diagram of the 2C expression construct design (see Materials and Methods). (B) The expressed 2C is a covalent heterodimer. On the left, SDS/PAGE analysis of Ni2+-agarose purified 2C. On the right, 2D nonequilibrium, pH-gradient electrophoresis. Both gels are Coomassie-stained. (C) The recombinant TCR 2C binds with high affinity to the Fab of the anti-idiotypic mAb 1B2 (21) using SPR on a BIAcore 2000 machine.
Figure 2
Figure 2
Purification and crystallization scheme for 2C TCR. Crystallization of 2C was achieved after many different purification protocols and covalent modifications were attempted. The final purification and carboxypeptidase digestion schemes that lead to high-resolution crystals are outlined in bold arrows.
Figure 3
Figure 3
Analysis of the purification and preparation steps of 2C for crystallization. (A) Ni2+-agarose-purified 2C was purified by gel filtration (Sephacryl-S-100) to remove aggregates and monomers. (B) Gel filtration-purified 2C was treated with carboxypeptidases A and B and purified by HIC. The progress of the reaction is monitored by HIC. Partially (Upper Left) and fully digested (Lower Left) 2C clearly separate by HIC and 2D nonequilibrium, pH-gradient electrophoresis (Upper Right). The fully digested 2C is purified by HIC and migrates on reducing and nonreducing SDS/PAGE at a position approximately 5 kDa smaller than the digested band (Lower Right).
Figure 4
Figure 4
Analysis of crystals of the 2C TCR. (A) Large crystals of 2C (B) SDS/PAGE analysis (silver-stained) of a washed 2C crystal reveals crystal is composed entirely of αβ covalent heterodimer. (C) IEF of a washed 2C crystal indicated that the predominant charge isoforms from the starting material are present in the crystallized protein. (D) Inspection of the crystal lattice packing of the 2C TCR crystals indicates that the α and β chain C termini of 2C are in contact with symmetry-related molecules.
Figure 5
Figure 5
Crystals of the 2C/H-2Kb-dEV8 complex. (A) A large, single crystal of 2C/H-2Kb-dEV8. (B) SDS/PAGE analysis of a washed crystal of 2C/H-2Kb-dEV8 reveals the presence of αβ TCR, Kb heavy chain, and β2m in stoichiometric amounts.
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