During the lifetime of a T cell, the αβ T cell receptor must recognize multiple different pMHC ligands in order for the cell to function. For example, the TCR recognizes a self-peptide bound to an MHC protein during selection in the thymus, thereby promoting T cell differentiation and migration to the peripheral lymphoid system. Subsequently, the TCR is capable of binding to a foreign antigenic peptide, most often associated with the same MHC protein, thereby driving the T cell to activation and elimination of the foreign agent. Finally, TCRs can bind to a foreign MHC protein (such as Ld in the 2C system) in situations that involve tissue transplants, thereby leading to transplant rejection (allore-actions).
To accomplish the recognition of different pMHC complexes, it has been proposed that TCRs exhibit flexibility in their CDRs, facilitating an “induced fit” mechanism of ligand binding (reviewed by Armstrong et al. (46
)). Recent studies, however, have suggested that structurally diverse TCRs can exhibit different thermodynamic binding mechanisms (13
). In the present report we have extended this observation to show that even a collection of structurally very similar TCRs, with as few as two amino acid substitutions, can exhibit diverse thermodynamic signatures for binding to the same pMHC ligand. By examining the reactions with the same pMHC ligand, we have avoided the possible influence of differences in the dynamics of the pMHC ligand that might account for the diverse TCR–pMHC binding thermodynamics.
In addition to examining the collection of TCRs against the same pMHC ligand, the structure of the unliganded pMHC (QL9-Ld
) was solved in order to assess whether the peptide undergoes significant structural changes upon TCR binding. Our results show that although there are minor adjustments of the peptide backbone and several side chains, there is no evidence of major structural reorganization. The side chain that appears to have the most mobility, as judged by a lack of electron density, was the phenylalanine at position 5 of QL9. We have shown previously that substitutions at this position can influence either Ld
binding or TCR binding, consistent with the possibility that side chain mobility and its position could vary depending on the nature of the side chain (37
). However, given the similar location of Phe5 in the 2C/QL9-Ld
, and m13/QL9-Ld
complexes, we suggest that even this position may not contribute appreciably to the differences in thermodynamics of binding by the TCRs. Nevertheless, it is clear from studies of different TCRs binding to the same pMHC that the side chain of a peptide residue can have quite divergent impacts on their binding energies (48
). Here, we show that the divergent impact can even extend to a collection of TCRs that are structurally very similar.
-m3 mutant was unique among the TCRs in that it exhibited favorable entropy (like wt 2C), an affinity increase associated largely with a faster on-rate, and enhanced specificity for the phenylalanine at QL9 position 5, relative to a tyrosine at this position. We have speculated that these effects may be related to an Arg31–Asp8 electrostatic interaction between the 1β
-m3 TCR and QL9 peptide, respectively. In this scenario, the electrostatic interaction could lead to both a faster on-rate and reduced entropic penalty associated with binding. The hydroxyl of the tyrosine at position 5 could be in sufficient proximity to influence this interaction. Alternatively, the thermodynamic binding signatures of these TCR mutants could be influenced, or even dominated, by a hydrophobic effect as has been observed in an elegant study of the role of a single tryptophan residue at the interface of the HEL antibody–lysozyme complex (49
). A recent comparative analysis of the structures of nine pairs of liganded and unliganded TCRs has shown that there is a hierarchy of conformational changes in different CDRs: 3α > 3β
> 1α > 2α > 2β
). Furthermore, their analysis suggested that conformational changes in CDRs in systems such as 2C are also associated with other changes at the interface, including electrostatic potential within the buried surface area of the TCR. Thus, it is especially difficult to attribute differences in thermodynamic signatures to any single mechanism, such as conformational effects, rather than to other differences at the interface (e.g., as indicated above, each of the mutants described here has at least one mutated residue that is charged). Additional studies of the dynamics of these interactions, using NMR or perhaps fluorescence-based approaches, will be required to more fully understand the mechanisms involved.
Finally, it is worth noting that CDR1, which is thought to have a less important role in peptide specificity than the CDR3s, actually exhibits the highest degree of specificity when comparing QL9 to the tyrosine 5 variant. This finding provides additional support to the notion that each of the CDRs is positioned to provide, directly or indirectly, a degree of peptide specificity that is the hallmark of T cell immunity.
Regardless of the mechanisms involved in discriminating structurally similar peptides, our results show that it is possible to engineer enhanced specificity into the interaction with peptide, as might be desirable in reactions with tumor antigens in order to avoid reactivity with a structurally similar self-antigen. Alternatively, we have shown with the high-affinity mutants 3α-m6 and 3α-m13 that one could engineer enhanced affinity against a peptide variant, as might be desirable for known viral escape variants that contain defined amino acid substitutions in key T cell epitopes.
In summary, this study shows that subtle changes in the structure of a single CDR can have profound effects on the thermodynamics and kinetics of TCR–pMHC interactions. These effects are accomplished without any significant alteration of the docking orientation or the footprint of the TCR on the QL9-Ld ligand. Subtle changes in the TCRs can also have a very significant effect on the fine specificity of the interactions, but the receptors retained biological activity as long as they were above a particular binding threshold.