Investigations into the Thermodynamic, Kinetic, and Structural Properties of TCR - peptide/MHC Interactions

The protein-protein recognition event that occurs at the interface between a T cell receptor (TCR) and a peptide presented by a major histocompatibility complex molecule (peptide/MHC) is essential to the adaptive immune system. This work examines TCR binding thermodynamics and compares these measurements to structural and functional features to gain insight into the determinants of TCR specificity, cross-reactivity, and binding affinity.

We have been studying the A6 TCR, which recognizes the Tax peptide presented by the class I MHC HLA-A2. We examined the A6-Tax/HLA-A2 complex formation via isothermal titration calorimetry (ITC) in order to study the thermodynamics of binding. However, binding caused a pKa shift in one or more ionizable groups, indicated by binding thermodynamics that varied with solution conditions. Calorimetric experiments with a variety of buffers, pH values, and temperatures were used to characterize the A6-Tax/HLA-A2 interaction. The data were fit globally to extract the intrinsic binding thermodynamic parameters. These parameters were compared to structural properties to help elucidate the basis for molecular recognition in this system.

Additionally, a high-affinity A6 TCR mutant, A6c134, binding to Tax/HLA-A2 was examined. The A6c134 variant has an approximately 800-fold stronger affinity for Tax/HLA-A2 as measured by SPR. This increase in affinity is due to a significantly slower off-rate for the A6c134-Tax/HLA-A2 complex than for the wild-type A6 counterpart. Crystallographically, the topology of the mutant and wild-type complex are similar, with small differences at the interface. Calorimetric data indicate that the A6c134 mutant binds Tax/HLA-A2 with opposing thermodynamics as compared to the wild-type interaction. Overall, the studies of A6 and A6c134 binding their cognate ligand, Tax/HLA-A2, have shown that each TCR binds with different thermodynamics and kinetics, despite maintaining similar binding footprints. Further studies on the movement of the CDR loops are proposed to give insight to the ground state of the TCR-pMHC interaction and the binding pathway behind the observed thermodynamic and kinetic parameters.