Multiple thermodynamic mechanisms contribute to the binding of pepMHC ligands by wild type and high-affinity TCRs (93.16)

Abstract

Abstract To better understand the mechanisms that govern TCR-pMHC binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from mouse CTL clone 2C, which recognizes QL9/Ld as a strong agonist. High affinity mutations of the 2C TCR were previously generated in various CDRs (1β, 2α, 3α and 3β) by in vitro directed evolution. Using a single-chain version of the TCR and a stabilized Ldprotein, we performed isothermal titration calorimetry (ITC) on these mutants, which bind with high affinity to QL9/Ld. Unexpectedly, our results showed that the wild-type interaction was favorable entropically. In contrast, all high affinity mutants except one (2C-1β) bound in an enthalpically driven manner. In addition, we measured the binding and kinetics of these mutants for both QL9/Ld and position 5 peptide variant Y5/Ld using SPR. While other mutants had higher affinity for the Y5 peptide variant than for QL9, the 2C-1β mutant had a lower affinity for Y5. Thus it appears that multiple thermodynamic mechanisms are used to achieve high affinity in this system. In addition, some high-affinity TCR mutants could achieve high affinity while exhibiting enhanced fine specificity. Supported by grants from the NIH.