Different thermodynamic binding mechanisms and peptide fine specificities associated with a panel of structurally similar high-affinity T cell receptors

Abstract

To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.

Figure 1

Summary of high-affinity mutant TCRs. (A) CDR mutations in various high-affinity 2C TCR mutants. The sequence of individual CDR loops in the wild-type 2C receptor are shown, with the sequence and name of any mutants isolated from degenerate libraries within the indicated CDR listed below. (B) SDS–PAGE showing purified scTCRs used in thermodynamic and kinetic studies. Each lane was loaded with 2 μg of purified protein. All scTCRs are near 30 kDa, as indicated by the molecular mass marker.

Figure 2

Thermodynamics of high-affinity scTCR binding to QL9-L^d. (A) Isothermal titration calorimetry (ITC) curves of 2C and high-affinity scTCRs titrated with QL9-L^d. (B) Summary of thermodynamic binding parameters associated with high-affinity scTCR/QL9-L^d interactions. The values for ΔG (solid white bars), ΔH (dark gray bars), and TΔS (light gray bars) for all interactions at 20 °C are shown in kcal/mol. ΔH and was determined by ITC, and ΔG and TΔS were calculated as described in the Experimental Procedures section. Error bars of ΔH represent the standard deviation over two or more independent experiments. Error bars for ΔG and TΔS indicate mathematically propagated error, based on error in the measured ΔH and K parameters.

Figure 3

Analysis of high-affinity scTCR binding to QL9-L^d and altered peptide ligand QL9-Y5-L^d. Surface plasmon resonance (SPR) traces of high-affinity scTCR binding to immobilized QL9-L^d (left) and QL9-Y5-L^d (right). Biotinylated QL9-L^d-m31 or QL9-Y5-L^d-m31 was immobilized on a streptavidin sensor chip surface (BIAcore) at 450–500 response units (RU). Soluble scTCRs were flowed over the surface at a flow rate of 30 μL/min at varying concentrations, depending on the affinity of the interaction. From top to bottom on immobilized QL9-L^d: [2C-T7], 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078 μM; [2α-m1], 10, 5, 2.5, 1.25, 0.625, and 0.078 μM; [3β-m4], 3.8, 1.9, 0.96, 0.48, 0.24, 0.12, 0.06, 0.03, and 0.015 μM; [3α-m13], 1.28, 0.64, 0.32, 0.16, 0.08, 0.02, and 0.01 μM; [1β-m3], 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, and 0.0195 μM; [3α-m6], 0.11, 0.053, 0.026, 0.013, 0.007, 0.003, 0.002, and 0.0008 μM. From top to bottom on immobilized QL9-Y5-L^d: [2C-T7], 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078 μM; [2α-m1], 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, and 0.04 μM; [3β-m4], 7.6, 3.8, 1.9, 0.96, 0.48, 0.24, 0.12, 0.06, and 0.03 μM; [3α-m13], 0.250, 0.063, 0.031, 0.016, 0.008, 0.004, and 0.002 μM; [1β-m3], 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, and 0.04 μM; [3α-m6], 0.105, 0.053, 0.026, 0.013, 0.007, 0.003, 0.002, and 0.0008 μM. To account for nonspecific interactions, binding to a biotin-blocked control surface was subtracted from the data. The bottom line in all traces represents an injection of buffer only.

Figure 4

Fine peptide specificity of high-affinity TCRs. Summary of kinetic binding affinities of scTCRs for QL9-L^d vs QL9-Y5-L^d. For mutants with higher affinity for QL9-L^d, fold difference in affinity was calculated as (affinity for QL9-Y5-L^d)/(affinity for QL9-L^d), and bars pointing down indicate the magnitude of the affinity difference. For mutants with higher affinity for QL9-Y5-L^d, fold difference in affinity was calculated as (affinity for QL9-L^d)/(affinity for QL9-Y5-L^d), and bars pointing up indicate the magnitude of the affinity difference. Error bars represent the standard deviation of two or more independent measurements of kinetic binding affinity by SPR.

Figure 5

Activation of high-affinity TCRs by QL9-L^d and QL9-Y5-L^d is CD8 independent. Percent maximal IL-2 release of CD8-negative TCR transfectants stimulated with (A) QL9-L^d or (B) QL9-Y5-L^d. Peptides were added to T2-L^d cells, and an equal number of T cell transfectant cells were added. Cells were incubated for 24 h at 37 °C, and supernatants were assayed for IL-2 using ELISA. % maximal IL-2 release = [(sample A₄₅₀ − null peptide A₄₅₀)/(CD3 A₄₅₀ − null peptide A₄₅₀)] × 100, where CD3 A₄₅₀ is the absorbance measured for cells stimulated with an anti-CD3 antibody and null peptide A₄₅₀ is the absorbance value for cells stimulated with a nullpeptide MCMV.

Figure 6

QL9 peptide does not undergo large conformational adjustments upon TCR binding. (A) Conformation of QL9 peptide in the structure of the QL9-L^d complex (red) with the simulated annealing omit map (cyan) contoured at 3.0σ. Because there was no adequate electron density for the side chain of the phenylalanine at position 5, an alanine residue was modeled at position 5 in the structure. (B) Comparison of the QL9 peptide from the unliganded QL9-L^d complex (red) and the 2C/QL9-L^d complex (blue). The positions of the TCR and MHC molecules are indicated with arrows.

Figure 7

Structures of TCR/QL9-L^d complexes. Superimposed structures of the 2C (blue), 3α-m6 (red), and 3α-m13 (cyan) TCRs in complex with QL9-L^d show similarities in (A) the overall structure of the complexes and (B) the CDR conformations and docking orientation on QL9-L^d. (C) Close-up of CDR3 loops in the 2C and high-affinity complexes. Residue 102 in the CDR3α is highlighted in all TCRs: S102 in 2C, R102 for m6, and P102 for m13. The phenylalanine residue at position 5 was replaced with a tyrosine using MacPyMOL software (DeLano Scientific LLC). (D) Close-up of the CDR1β loop in the 2C/QL9-L^d complex. The 2C TCR and the position of the two affinity mutations in the 1β-m3 mutant are shown (N28G and N31R) in blue. In all structures, QL9 is shown in magenta and L^d is shown in brown.