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A computer-generated model of the single-chain variable V alpha V beta fragment of the RFL3.8 T-cell receptor (TCR) specific for fluorescein served as a starting point for mutagenesis aimed at identification of its antigen-contacting residues. Selected backbone segments of the model representing regions of prominent sequence similarity between antibodies and TCRs were least-squares superimposed onto the corresponding segments of the crystallographically resolved 4-4-20 antibody complexed with its antigen, fluorescein. The superimposition placed the antibody-bound fluorescein molecule close to a cavity on the surface of the TCR model formed by the complementarity-determining region (CDR) loops. Some of the TCR cavity forming loops displayed sequence motifs related to canonical CDR loops previously found in antibodies. Six putative amino acid contacts were identified and single-chain TCRs with mutations at each of these positions were expressed in Escherichia coli, purified, refolded, and assayed for fluorescein binding. Five of the six mutations resulted in a loss of detectable binding. These RFL3.8 antigen combining site residues are distributed among the beta 3, alpha 1, and alpha 2 CDR loops and show striking chemical similarity to the known fluorescein contact residues on 4-4-20. Thus, antibodies and TCRs are similar both in their overall architecture and in the chemical details of specific antigen recognition.
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