A conserved energetic footprint underpins recognition of human leukocyte antigen-E by two distinct αβ T cell receptors

Abstract

αβ T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8⁺ T cell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8⁺ T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of a TRBV9⁺ TCR in complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9⁺ TCR adopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14⁺ TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells.

Keywords: T-cell receptor (TCR); major histocompatibility complex (MHC); mutagenesis; receptor structure-function; structural biology; viral immunology.

Figure 1.

Representative sensorgrams showing the binding of GF4 TCR to peptides presented by HLA-E by surface plasmon resonance. GF4 was captured on the surface of a Bio-Rad ProteOn GLC chip by anti-TCR mAb 12H8 (25) and assessed for its ability to interact with HLA-E presenting LIL or LVL (A and B, respectively). Increasing concentrations of HLA-E (A, 100, 40, 16, 6.4, and 2.6 μm; B, 30, 12, 4.8, 1.9, and 0.8 μm) were passed over captured GF4 TCR. K_D was determined by equilibrium analysis (right panels) and by kinetic analysis for VMAPRTLVL (dashed lines indicate data fit). Data show representative sensorgrams of at least three experiments with independent preparations of refolded proteins.

Figure 2.

A and B, the structure of the HLA-E (yellow schematic) presenting either LVL (A) or LIL (B) peptide (black sticks) to GF4 TCR (α-chain in pink schematic for LIL and β-chain in blue schematic); β₂-microglobulin (β₂m) is represented in gray schematic. C and D, structural footprint of the GF4 TCR onto HLA-E–LVL (C) or HLA-E–LIL (D). The contribution of the CDR loops to the BSA of pHLA-E is represented for GF4 HLA-E–LVL (C) and HLA-E–LIL (D) complexes. The HLA-E atoms making contacts with CDR1α (teal), CDR2α (green), CDR3α (purple), framework α (pink), CDR1β (red), CDR2β (orange), CDR3β (yellow), or framework β (blue) are colored accordingly to the TCR segment contacted, whereas the magenta and blue spheres represent the center of mass for the Vα and Vβ, respectively. The percent contribution of the CDR loops and framework regions of the GF4 TCR–HLA-E–LVL (C) and GF4 TCR–HLA-E–LIL (D) complexes in binding to the pHLA-E complex is also shown in the pie charts.

Figure 3.

A–D, GF4 TCR β-chain interactions with HLA-E (white) via the CDR1β (red) and CDR3β residues (yellow) (A) via the CDR2β (orange) framework β (pale blue) (B, C, and D). E and F, GF4 TCR α-chain interactions with HLA-E α2 helix via the CDR1α (teal), CDR2α (green), and framework α (pale pink) (E) as well as via the CDR3α (purple) (F). Residues interacting are depicted as sticks, hydrophobic bonds are shown as blue dashed lines, and salt bridges or hydrogen bonds are shown as red dashed lines.

Figure 4.

A–C, GF4 TCR interactions with the peptides: LVL peptide (black stick) interactions with CDR3β (yellow) (A) and CDR3α (purple) (B). Superimposition of GF4 TCR CDR3 loops in complex with the HLA-E–LIL and HLA-E–LVL is shown in C with the CDR3α loops in pink, CDR3β loops in blue for the GF4 TCR–HLA-E–LVL and lighter shade for the GF4 TCR–HLA-E–LIL, and the peptides in black (LVL) and gray (LIL). Residues interacting are depicted as sticks, hydrophobic bonds are shown as blue dashed lines, and salt bridges are shown as red dashed lines. D, superimposition of KK50.4 TCR–HLA-E–LIL (green schematic) and GF4 TCR–HLA-E–LVL (purple schematic). E and F, structural footprint of KK50.4 TCR onto HLA-E–LIL (E) and GF4 TCR onto HLA-E–LVL (F). The pHLA-E atoms making contacts with each TCR are shown in light pink (α-chain) and blue (β-chain). The magenta and blue spheres represent the center of mass for the Vα and Vβ, respectively.

Figure 5.

Superimposition of the KK50.4 TCR–HLA-E–LIL (green) and GF4 TCR–HLA-E–LVL (purple) structures with the CDR3α loops surrounding the P4-P5 peptide residues (A) and the CDR3 loops around the P8 position of the peptides (B) is shown. C shows a superposition of the KK50.4 (green) and GF4 (purple) TCR CDR2β loops and their interactions with the HLA-E molecule; the lighter shade-colored residues are from the framework β segment. The dashed lines indicate the interactions between residues in each complex. Affinity of HLA-E mutants to KK50.4 TCR (D) or GF4 TCR (E) is represented as relative percentage of the wild-type (WT) value determined by surface plasmon resonance as well as the energetic footprints of each TCR on HLA-E. Dotted lines represent 30% (or 3 times binding reduction) in orange and 20% (or 5 times binding reduction) in red of the binding affinity compared with WT HLA-E. The effect of each mutation (yellow, no effect; orange, 3 times binding reduction; red, 5 times binding reduction) is represented on the HLA-E surface. Peptides residues are shown in gray. Error bars represent S.E.