T cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-MHC molecular flexibility

Abstract

T cell-mediated immunity requires T cell receptor (TCR) cross-reactivity, the mechanisms behind which remain incompletely elucidated. The alphabeta TCR A6 recognizes both the Tax (LLFGYPVYV) and Tel1p (MLWGYLQYV) peptides presented by the human class I MHC molecule HLA-A2. Here we found that although the two ligands are ideal structural mimics, they form substantially different interfaces with A6, with conformational differences in the peptide, the TCR, and unexpectedly, the MHC molecule. The differences between the Tax and Tel1p ternary complexes could not be predicted from the free peptide-MHC structures and are inconsistent with a traditional induced-fit mechanism. Instead, the differences were attributable to peptide and MHC molecular motion present in Tel1p-HLA-A2 but absent in Tax-HLA-A2. Differential "tuning" of the dynamic properties of HLA-A2 by the Tax and Tel1p peptides thus facilitates cross-recognition and impacts how structural diversity can be presented to and accommodated by receptors of the immune system.

Figure 1

The unligated Tel1p and Tax complexes with HLA-A2 are sequence and structural mimics. A) Sequences of the Tax and Tel1p peptides. B) Structure of the unligated Tel1p-HLA-A2 peptide-binding domain superimposed on the Tax-HLA-A2 peptide binding domain. Superimposition is through the backbones of the peptide-binding domains. The color code is given at the bottom of the figure and maintained in panels C and D. C) Superimposition of the peptides from the free Tel1p-HLA-A2 structure and the Tax peptide from the free Tax-HLA-A2 structure. D) Superimposition of the peptides from the free Tel1p-HLA-A2 structure and the Tax peptide from the A6–Tax-HLA-A2 structure. Superimposition for all panels is by backbone atoms.

Figure 2

Recognition of Tel1p-HLA-A2 by the A6 TCR proceeds with changes in the peptide, the TCR, and the HLA-A2 α2 helix. The color code for all panels is given in the lower right of the figure. A) Overview of the A6–Tel1p-HLA-A2 and A6–Tax-HLA-A2 complexes. B) Cross-eyed stereo view highlighting the three major changes in the interface: peptide, TCR CDR3β, and HLA-A2 α2 helix. C) Close-up view of the changes that occur in the Tel1p peptide upon TCR binding. The rotations of the Tyr5 and Gln7 side-chains are indicated, as is the distance moved and the compensating rotations in the Tyr5 ψ and the Leu6 φ bonds. The clash that would occur between the Tyr5 side-chain and the peptide backbone if the backbone did not rotate is indicated in red. D) Close-up of the difference in the HLA-A2 α2 helix between the Tel1p and Tax complexes with A6. The differential position of the Ala150 carbon in the two complexes is indicated, and the shifting region of the helix is in red. E) Close-up of the difference in the A6 CDR3β loop between the Tel1p and Tax complexes with A6. The differential position of the carbon of Gly101β in the two complexes is indicated. F) The various conformational changes are mutually dependent. Steric clashes occur if the Tel1p peptide from the Tel1p ternary complex is fit into the peptide-binding domain from the unligated Tel1p-HLA-A2 complex or into the A6–Tax-HLA-A2 interface. Clashes, defined as interatomic distances less than the sum of the corresponding van der Waals radii, are shown as dashed red lines. Superimposition for all panels is through backbone atoms of the variable and peptide-binding domains.

Figure 3

Recognition of Tel1p by A6 proceeds with changes in the CDR3β loop, but other loops are largely unaltered. A) Cross-eyed stereo view of the CDR loop positions in the complexes of A6 with the Tel1p, Tax, and Tax-5K-IBA peptides (color code is under the panel). The view is through the peptide-binding domain of HLA-A2, with the Tel1p peptide shown in magenta for reference. Compared to recognition of the Tax peptide, the large shift in CDR3β upon recognition of Tel1p is evident, and it is similar but slightly smaller than the shift seen in recognition of Tax-5K-IBA (Gagnon et al., 2006). B) Comparison of the TCR central pocket in the Tel1p (left) and Tax (right) ternary complexes. The view is as in panel A, with the surface of the TCR shown. CDR3β is blue and CDR3α is yellow. Superimposition for both panels is by the backbones of the variable domains.

Figure 4

Proline mutagenesis verifies the switch that occurs in the HLA-A2 α2 helix upon A6 recognition of the Tel1p peptide and suggests coupling between motion in the α2 helix and the Tel1p peptide. A) Proline at position 150 would break the usual HLA-A2 α2 helix conformation, as a hydrogen bond is lost and interatomic bumps are introduced (left; additional atoms from the proline ring are yellow). In contrast, the altered conformation seen in A6 recognition of the Tel1p peptide can tolerate aproline at position 150 (right). B) Results for the A6 TCR binding wild type or A150P peptide-HLA-A2 presenting the Tax peptide (left) or the Tel1p peptide (right). Consistent with the structural data, the A150P mutation weakens affinity for Tax but enhances affinity for Tel1p. C) The structures of the A150P HLA-A2 mutant presenting the Tel1p and Tax peptide verify the mutation stabilizes the altered conformation. The “normal” α2 helix conformation from the unligated Tel1p complex is grey, the conformation from the A6–Tel1p-HLA-A2 ternary complex is cyan, the conformation from the unligated A150P mutant with Tel1p is brown, and the conformation from the unligated A150P mutant with Tax is burgundy. Superimposition is via backbone atoms of the peptide-binding domains. D) Electron density for peptide side chains in the A150P Tel1p structure (top) is weaker than the density in the A150P Tax structure (bottom), despite the fact that the Tel1p structure is of slightly better resolution. This suggests a coupling between mobility in the Tel1p peptide and mobility in the HLA-A2 α2 helix. Densities are 2F_o-F_c contoured at 1σ.

Figure 5

The HLA-A2 α2 helix linker region is more dynamic when the Tel1p rather than the Tax peptide is presented. A) Steady-state anisotropies for Tel1p-HLA-A2 fluoresce in (Flc) labeled at position 151 are 25% lower than those for Tax-HLA-A2 labeled at position 151, indicating greater backbone flexibility in the Tel1p complex. The position 151 values are substantially higher than those for protein labeled at position 195, located in a loop at the tip of the α3 domain. Anisotropies for α2 helix positions 145 and 138, located outside of the linker region, are independent of which peptide is bound. Similar results were seen for position 151 when labeled with BODIPY-FL. Data shown are averages and standard deviations of 50 measurements performed at 25 °C on three independently generated samples at 100 nM. B) The differential dynamics of the HLA-A2 α2 helix revealed by steady-state anisotropy can be attributed to increased fluctuations occurring over the nanosecond timescale. In a time resolved experiment, 50% of the initial anisotropy at position 151 was lost by 8.4 ns for the Tax complex and 3.5 ns for the Tel1p complex. For comparison, position 195 lost 50% of its initial anisotropy in 1.1 ns.

Figure 6

Molecular dynamics simulations of free pMHC complexes confirm the greater dynamics present in the Tel1p-HLA-A2 complex. A) Amino acids in the α2 helix linker region sample a range of ψ, φ bond angles in the Tel1p simulation, including both the TCR-free and TCR-bound conformations for Ala149. These positions in the Tax simulation are more static. B) Tyr5 and Gln7 sample both TCR-free and TCR-bound conformations in the Tel1p simulation. C) In the Tel1p simulation, the dynamic cross-correlation matrix reveals correlated motion between the C-terminal half of the peptide and the HLA-A2 α2 helix linker region. Little or no correlations are seen between the peptide and α2 helix in the Tax simulation.