Energetic Impact of Individual Peptide Residues on Binding by High-Affinity TCRs: Implications for Antigen Specificity (93.17)

Abstract

Abstract The mouse CD8+ clone 2C recognizes the syngeneic pMHC ligand SIYR:Kb and the allogeneic pMHC ligand QL9:Ld. High-affinity mutants of the 2C TCR have been generated previously against these ligands by directed evolution using yeast display. In order to generate specific probes for the detection of these pMHC ligands, we have developed a strategy to express high-affinity TCRs that are biotinylated in vivo in E. coli. The biotinylated TCRs and a collection of single amino acid variants of peptides SIYR and QL9 were used to quantitate relative energy contributions of each peptide residue to TCR binding. Generation of a quantitative binding energy map allowed comparison of two structurally very different MHC (Kb versus Ld) that bind to the same TCR. The energetic contributions of different residues from the peptides SIYR and QL9 were distinct. Analysis of QL9:Ld showed that four central positions of peptide QL9 contributed a significant amount of binding free energy to both wild type and high-affinity TCRs. Analysis of SIYR:Kb showed that there are distinct differences, compared to QL9:Ld, in recognition by the same family of mutated TCRs. Using crystal structures of TCR/QL9:Ld and TCR/SIYR:Kb complexes, it was possible to assess whether binding energies were influenced directly through contacts with the TCR, or indirectly through peptide induced conformational changes in the peptide or MHC. Supported by grants from the NIH.