Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes

Abstract

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8⁺ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

Keywords: CD8 T cells; CP: Immunology; HLA-E; MHC Ia; MHC-E; NK cells; NKG2A; SAXS; T cell receptor; VL9; X-ray crystallography; small-angle X-ray scatter.

Graphical abstract

Figure 1

SEC-SAXS analysis of peptide-HLA-E complexes (A) (i), (iii), (v), (vii), (ix), and (xi): Log₁₀ scattering intensity plots for HLA-E SEC-SAXS experiments. Scattering intensity curves for HLA-E refolds run in the absence or presence of 120 μM excess peptide during elution are plotted. On the x axis is the scattering vector q (Å⁻¹) versus the scattered intensity, I(q), log-scale, on the y axis. (ii), (iv), (vi), (viii), (x), and (xii): Normalized Kratky plots with superimposed curves corresponding to HLA-E SEC-SAXS experiments with or without 120 μM excess peptide. Modulated Gaussian curves are color-coded according to legends in adjacent log₁₀ intensity plots. The scattering vector multiplied by the radius of gyration is plotted on the x axis versus the scattering intensity I(q) divided by the experiment’s I(0) multiplied by (q^∗R_g)² on the y axis. (B) Ab initio DAMMIF models (small dots) represent the average conformational protein state in solution. Top: SEC-SAXS runs where HLA-E complexes were injected onto the HPLC column in the presence of 120 μM excess peptide but without excess peptide during elution. Bottom: 120 μM excess peptide was present throughout injection and elution. HLA-E-peptide structural coordinates superimposed via SUPCOMB onto their peptide-equivalent SEC-SAXS-based bead models are reported for VL9 (PDB: 1MHE), Mtb44 HLA-Mtb44 (PDB: 6GH1), IL9 (structure reported here), Mtb14 (structure reported here) and RL9HIV (PDB: 6LH1).

Figure 2

SEC-SAXS analysis for peptide-HLA-A2 complexes (A) (i) and (iii): Log₁₀ scattering intensity plots for HLA-A^∗02:01 SEC-SAXS experiments. The scattering vector q (Å⁻¹) is plotted (x axis) versus scattered intensity, I(q), log scale (y axis). Scattering intensity curves for HLA-A2 refolds run with or without 120 μM excess peptide during elution, are plotted. (ii) and (iv): Normalized Kratky plots with superimposed curves corresponding to HLA-A2 SEC-SAXS experiments with or without 120 μM excess peptide. Modulated Gaussian curves are color-coded according to figure legends in log₁₀ intensity plots. The scattering vector multiplied by the radius of gyration is plotted (x axis) versus the scattering intensity I(q) divided by the experiment’s I(0) and multiplied by (q^∗R_g)² (y axis). (B) HLA-A^∗02:01-peptide refolds tested via SEC-SAXS. Each row represents SEC-SAXS experiments conducted in the absence or presence of 120 μM excess peptide. The radius of gyration (R_g) and maximum dimension (d_max), both measured in Å, are specified, along with circular dichroism-measured T_m values (Borbulevych et al., 2009).

Figure 3

DAMMIF model structural alignment negatively correlates with HLA-E thermal stability and peptide-binding signals (A) (i–iv): Scatter graphs where ELISA-derived HLA-E peptide-binding signals or T_m values are plotted on the x axis versus SEC-SAXS-obtained d_max values or molecular envelope volumes (denoted in Å), on the y axes. (v): Scatter graph with normalized ELISA-based HLA-E peptide-binding signals and T_m, denoted in A, plotted on the x and y axes, respectively. (vi): Figure legend for A (i–v). (B) (i–vii): Bar charts representing ELISA-based HLA-E peptide titration assays, with peptide concentration (x axis) versus average absorbance signals at 450 nm (y axis). The “peptide-free” negative control (dark gray) reflects the “no-rescue” exchange reaction. For (ii–vii), the positive control VL9 (VMAPRTVLL) peptide (dark red) corresponds to a 120 μM excess peptide exchange reaction. For (i–vii), Pearson’s correlation coefficients (r) are denoted, with corresponding p values or ns for non-significant correlations. Error bars depict SEM. Biological repeats, n = 3; technical replica n = 2.

Figure 4

Structural characterization of mycobacterial peptide binding to HLA-E (A) (i): PyMol visualization (side-on) of peptide Mtb14, from molecule 1 of the asymmetric unit (hot pink stick form). An electron density map contoured to 1 sigma is displayed (gray mesh) overlaying the peptide. (ii): Ribbon representation of the peptide backbone (hot pink) from the HLA-E-Mtb14 structure with a superimposed VL9 peptide backbone (PDB: 1MHE, violet). The peptide N and C termini are labeled for clarity. (B) (i): Side-on visualization of peptide IL9, from molecule 1 of the asymmetric unit (blue stick form). (ii): Side-on visualization of peptide IL9 from molecule 2 of the asymmetric unit (blue stick form). In B (i) and (ii), an electron density map contoured to 1 sigma is displayed (gray mesh) overlaying the peptide. (iii): Superimposed peptide backbones are depicted with the N and C termini labeled. The IL9 peptide backbones from molecules 1 and 2 of the asymmetric unit (blue ribbon) with the position 7 A side chains (blue stick form) are reported. The distance (1.9 Å) separating the position 7 Cα atom of the IL9 peptides from molecules 1 and 2 of the asymmetric unit is denoted. (iv): Superimposed peptide backbones are depicted in ribbon form with the N and C termini labeled. The IL9 peptide backbones from molecules 1 and 2 of the asymmetric unit are displayed (blue), with the VL9 peptide backbone from the published HLA-E-VL9 structure (PDB: 1MHE) in violet. The 2.0 Å distance separating the IL9 position 7 Cα atom from molecule 1 of the asymmetric unit and the VL9 position 7 Cα atom is denoted.

Figure 5

Suboptimal E pocket interactions compromise HLA-E complex stability (A) (i): HLA-A2 E pocket (PDB: 3MRG [Reiser et al., 2014]) visualization with side chains of pocket-forming residues depicted (gray or green sticks). The hepatitis C virus-derived CINGVCWTV peptide (pink ribbon) with the solvent-exposed position 7 side chain (stick form) projecting away from the E pocket. HLA-A2 groove regions including the α2 helix and β-sheet floor are shown (gray cartoon). (ii–v): Position 7 anchor side-chain-accommodating E pocket of HLA-E. E-pocket-forming residue side chains are depicted (gray sticks) with remaining regions of the PBG (gray cartoon). In (ii), the VL9 peptide main chain (purple ribbon) with the position 7 side chain (purple stick-form) projecting downward into the secondary E pocket is shown (PDB: 1MHE). (iii–v): Illustration of differential positioning at position 7 of HLA-E-bound pathogen-derived peptides relative to VL9 (PDB: 1MHE). The IL9, RL9HIV, and Mtb14 peptide backbones are depicted (blue [iii], yellow [iv], and magenta [v] ribbons, respectively), with position 7 side chains: Ala-7 of IL9, Ser-7 of RL9HIV, and Gln-7 of Mtb14 (blue [iii], yellow [iv], and magenta [v] stick form, respectively). The superimposed VL9 peptide main chain (PDB: 1MHE) is shown (purple ribbon), and the distance between the aligned peptide position 7 Cα atoms is indicated by dashed lines for the following: 2 Å for VL9 and IL9, 3.4 Å for VL9 and RL9HIV, and 1.1 Å for VL9 and Mtb14. In (v), water-mediated inter-chain hydrogen bonds (magenta dashed lines) with the coordinated H₂O molecule (cyan-shaded sphere) are depicted. Four hydrogen bonds, which indirectly link the Mtb14 position 7 Gln side chain to the HLA-E α2-helix Ser-147 side chain and Ser-143 main chain, in addition to the Mtb14 peptide position 8 main chain, are shown. The Mtb14 Val-8 main chain and HLA-E Ser-143 main chain are labeled “MC” (magenta and gray sticks, respectively). (B) HLA-E-β2M-peptide thermal stability (T_m) in the presence of 12 M excess peptide. T_m values (°C) are listed for VL9, Mtb14, and the corresponding position (p)7 variant peptides VL9 p7Q and Mtb14 p7V. (C) (i): Bar chart of an ELISA-based HLA-E peptide-binding assay in which the wild-type Mtb14 peptide was compared with the Mtb14 p7V peptide. The VL9 peptide-positive control (dark red) and the negative peptide-free “no rescue” control (gray) are indicated. Test peptides are plotted (x axis) versus the average absorbance readings at 450 nm (y axis); p values from unpaired t tests are denoted no asterisk p > 0.05, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001. Error bars depict SEM. Biological repeats, n = 3; technical replica, n = 2. (ii): Bar chart as described in (C) (i), in which HLA-E binding to VL9 was compared with a variant peptide containing p7Q. The Mtb14 peptide, which naturally contains p7Q, was included for reference. (D) (i) and (iii): Log₁₀ scattering intensity plots for HLA-E SEC-SAXS experiments. Plotted on the x axis is the scattering vector q (Å⁻¹) with scattered intensity, I(q), log scale on the y axis. Superimposed scattering intensity curves for peptide-HLA-E refolds are color coded according to the corresponding figure legend. (ii) and (iv): Normalized Kratky plots with superimposed curves from HLA-E SEC-SAXS experiments. Superimposed modulated Gaussian curves are color coded according to legends in log₁₀ intensity plots. The scattering vector multiplied by the radius of gyration (x axis) is plotted versus the scattering intensity I(q) divided by the experiment’s I(0) and multiplied by (q^∗R_g)² (y axis). (E) Summary detailing HLA-E-peptide refolds tested via SEC-SAXS. Peptide ID, sequence and origin are specified along with the radius of gyration (R_g) and maximum dimension (d_max), both measured in Å.

Figure 6

Distinct structural motifs emerge in the absence of HLA-E-bound VL9 peptide (A) (i): Superimposed HLA-E α2 helical kink regions depicted as lines with the α2 helix short-arm labeled “SA,” the long-arm labeled “LA,” and residue positions denoted. The HLA-E-VL9 α2 helix (PDB: 1MHE) is shaded gray. Superimposed pathogen peptide-bound HLA-E α2 helices are depicted, including HLA-E-Mtb44 (green), HLA-E-RL9HIV (yellow), HLA-E-Mtb14 (magenta), and HLA-E-IL9 (blue). (ii): The maximum distance in Å separating Cα atoms of HLA-E-VL9 α2 helix residues (1MHE) versus corresponding Cα atoms in pathogen peptide-bound HLA-E structures. (B) Aligned HLA-E-bound peptide Cα backbones are depicted as ribbons following whole HLA-E complex superposition. The VL9 peptide (PDB: 1MHE) is shaded gray. Pathogen-derived HLA-E-bound peptides are colored green (Mtb44), yellow (RL9HIV), magenta (Mtb14), and blue (IL9). HLA-E α1 and α2 helices plus N and C peptide termini positions are indicated. Position 5 Cα atoms are circled, and the maximum distance separating the VL9 peptide Arg-5 Cα from pathogen peptide position 5 Cα atoms are denoted and measure 2.3 Å for VL9 and RL9HIV, 1.9 Å for VL9 and IL9, 1.9 Å for V9 and Mtb14, and 1.5 Å for VL9 and Mtb44. (C) (i): Visualization of the HLA-E α2 helix (gray cartoon) with aligned pathogen-derived peptide backbones RL9HIV, IL9, and Mtb14, in yellow, blue, and magenta ribbon, respectively. The superimposed VL9 peptide backbone (PDB: 1MHE) is shown in gray ribbon. The two salt bridges connecting HLA-E Glu-152 and the Arg-5 side chain of the VL9 peptide are shown (gray dashed lines). The HLA-E Gln-156 side chain-VL9 Arg-5 oxygen main-chain hydrogen bond is also depicted (gray dashed lines). (ii): The HLA-E α2 helix is shown (gray cartoon) with the aligned Mtb44 (green) and VL9 (PBD: 1MHE[gray]) peptide backbones. Molecule 3 of the HLA-E-Mtb44 structure, in which a hydrogen bond (green dashed line) connects the HLA-E Gln-156 side chain to the main chain oxygen of Mtb44 Lys-5 (green sticks) is depicted. This hydrogen bond is present in one of the four molecules in the asymmetric unit. For C (i) and (ii), HLA-E α2 helix Ser-147, Glu-152, and Gln-156 side chains (stick form) are color-coded according to the corresponding peptide. (D) The HLA-E α2 helix is shown (gray cartoon) with the aligned RL9HIV and IL9 peptides (yellow and blue ribbon, respectively). The RL9HIV and IL9 Tyr-3 side chains are shown in yellow and blue stick form, respectively. HLA-E α2 helix Glu-152 side chains from the HLA-E-RL9HIV and HLA-E-IL9 structures are shown (yellow and blue sticks, respectively) with corresponding hydrogen bonds (yellow/blue dashed lines). The HLA-E-VL9 Glu-152 side chain (PBD : 1MHE) is shown in gray stick form for reference. (E) Details of published HLA-E-peptide structures plus the 2 HLA-E structures presented here. Peptide IDs, origins, and amino acid sequences are specified with the corresponding HLA-E allotype and PDB accession codes.