Peptide-MHC Binding Reveals Conserved Allosteric Sites in MHC Class I- and Class II-Restricted T Cell Receptors (TCRs)

Abstract

T cells are vital for adaptive immune responses that protect against pathogens and cancers. The T cell receptor (TCR)-CD3 complex comprises a diverse αβ TCR heterodimer in noncovalent association with three invariant CD3 dimers. The TCR is responsible for recognizing antigenic peptides bound to MHC molecules (pMHC), while the CD3 dimers relay activation signals to the T cell. However, the mechanisms by which TCR engagement by pMHC is transmitted to CD3 remain mysterious, although there is growing evidence that mechanosensing and allostery both play a role. Here, we carried out NMR analysis of a human autoimmune TCR (MS2-3C8) that recognizes a self-peptide from myelin basic protein presented by the MHC class II molecule HLA-DR4. We observed pMHC-induced NMR signal perturbations in MS2-3C8 that indicate long-range effects on TCR β chain conformation and dynamics. Our results demonstrate that, in addition to expected changes in the NMR resonances of pMHC-contacting residues, perturbations extend to the Vβ/Vα, Vβ/Cβ, and Cβ/Cα interfacial regions. Moreover, the pattern of long-range perturbations is similar to that detected previously in the β chains of two MHC class I-restricted TCRs, thereby revealing a common allosteric pathway among three unrelated TCRs. Molecular dynamics (MD) simulations predict similar pMHC-induced effects. Taken together, our results demonstrate that pMHC binding induces long-range allosteric changes in the TCR β chain at conserved sites in both representative MHC class I- and class II-restricted TCRs, and that these sites may play a role in the transmission of signaling information.

Keywords: Allostery; NMR; T cell receptor; T cell triggering; peptide–MHC.

Figure 1:

Model of the TCR–CD3 complex with bound pMHC ligand. The model was constructed by superposing the crystal structure of the MS2–3C8–MBP–HLA-DR4 complex (PDB 3O6F) [23] onto the cryoEM structure of the unbound TCR–CD3 complex (PDB 6JXR) [2] through the shared Cα/Cβ domains. The inset shows a close-up view of the Cβ FG loop in the TCR–CD3 complex. The Cβ FG loop is positioned immediately above CD3εγ. The side chains of Cβ FG loop residues that undergo NMR signal perturbation in the MS2–3C8(wt)–MBP–HLA-DR4 complex (see Results) are drawn in stick representation.

Figure 2:

Variants of the MS2–3C8 TCR ectodomain used in this study. (A) Crystal structure of MS2–3C8 (PBD 3O6F) [23] highlighting amino acid positions on the α (green) and β chains (wheat) where mutations were made. (B) Amino acid sequence comparisons between the three MS2–3C8 variants, MS2–3C8(mut1), MS2–3C8(mut2), and MS2–3C8(wt).

Figure 3:

NMR backbone assignment of MS2–3C8 TCR β chains. (A) Overlaid two dimensional ¹H-¹⁵N TROSY-HSQC spectra for MS2–3C8(mut1)α[β−²H, ¹⁵N] (black), MS2–3C8(mut2)α[β−²H, ¹⁵N] (green), and MS2–3C8(wt)α[β−²H, ¹⁵N] (red). Unassigned main chain signals are marked (x). (B) Expanded region highlighted (dashed box) in (A).

Figure 4:

Summary of backbone amide CSPs and differential peak intensity changes in the complex between MS2–3C8(mut1)α[β−²H, ¹⁵N] and MBP–HLA-DR4. (A) Expanded regions from the superimposed two dimensional ¹H-¹⁵N TROSY-HSQC spectra of unbound (black) and MBP–HLA-DR4-bound (red) states of MS2–3C8(mut1)α[β−²H, ¹⁵N]. The spectrum of the bound state is scaled to match the peak intensities of most signals in the unbound state. This is done for visual purposes only to highlight the disproportional changes for specific residues upon binding. (B) Combined ¹H and ¹⁵N chemical shift perturbations, Δδ_total (ppm), in the MS2–3C8(mut1) β chain versus residue number. The dashed line represents the mean value plus 1SD. Gray histogram bars indicate unassigned and proline residues. (C) Plot of percent loss of backbone amide peak intensity versus residue number. The dashed line corresponds with the mean plus 1SD. Hypervariable regions, αA, and the Cβ FG loop are highlighted in (B) and (C). (D) β chain residues in MS2–3C8(mut1) with experimentally significant changes (≥mean plus 1SD) upon binding to MBP–HLA-DR4: MS2–3C8(mut1) β chain (wheat); CSPs (blue); peak intensity changes (red); both CSP and peak intensity (purple); unassigned residues (gray); MS2–3C8(mut1) α chain (green); MBP peptide from pMHC (orange).

Figure 5:

Summary of backbone amide CSPs and differential peak intensity changes in the complex between MS2–3C8(mut2)α[β−²H, ¹⁵N] and MBP–HLA-DR4. (A) Expanded regions from the superimposed two dimensional ¹H-¹⁵N TROSY-HSQC spectra of unbound (black) and MBP–HLA-DR4-bound (red) states of MS2–3C8(mut2)α[β−²H, ¹⁵N]. The spectrum of the bound state is scaled as in Fig. 4A. (B) Combined ¹H and ¹⁵N chemical shift perturbations, Δδ_total (ppm), in the MS2–3C8(mut2) β chain versus residue number. The dashed line indicates the mean value plus 1SD. Gray histogram bars indicate unassigned and proline residues. (C) Plot of percent loss of backbone amide peak intensity versus residue number. The dashed line corresponds with the mean plus 1SD. Hypervariable regions, αA, and the Cβ FG loop are highlighted in (B) and (C). (D) β chain residues in MS2–3C8(mut2) with experimentally significant changes (≥mean plus 1SD) upon binding to MBP–HLA-DR4. Color coding as in Fig. 4D.

Figure 6:

Summary of backbone amide CSPs and differential peak intensity changes in the complex between MS2–3C8(wt)α[β−²H, ¹⁵N] and MBP–HLA-DR4. (A) Expanded regions from the superimposed two dimensional ¹H-¹⁵N TROSY-HSQC spectra of unbound (black) and MBP–HLA-DR4-bound (red) states of MS2–3C8(wt)α[β−²H, ¹⁵N]. The spectrum of the bound state is scaled as in Figs. 4A and 5A. (B) Combined ¹H and ¹⁵N chemical shift perturbations, Δδ_total (ppm), in the MS2–3C8(wt) β chain versus residue number. The dashed line corresponds with the mean value plus 1SD. Gray histogram bars indicate unassigned and proline residues. (C) Plot of percent loss of backbone amide peak intensity versus residue number. The dashed line represents the mean plus 1SD. Hypervariable regions, αA, and the Cβ FG loop are highlighted in (B) and (C). (D) β chain residues in MS2–3C8(wt) with experimentally significant changes (≥mean plus 1SD) upon binding to MBP–HLA-DR4. Color coding as in Figs. 4D and 5D.

Figure 7:

Comparison of changes in MHC class II-restricted TCR MS2–3C8 and MHC class I-restricted TCR A6 β chains upon pMHC-binding. (A-D) Summary of conserved and consensus MS2–3C8 residues perturbed by binding to the MBP–HLA-DR4. Color-coding for conserved residue perturbations present in all three MS2–3C8 variants: CSPs (blue); peak intensity changes (red); both CSP and peak intensity (purple). Color-coding for consensus residue perturbations: CSPs (cyan); peak intensity (orange); both CSP and peak intensity (light purple). (E-H) TCR A6 residues perturbed by binding to Tax–HLA-A2. Color-coding: CSPs (blue); peak intensity changes (red). Additional color coding: β chain (wheat); α chain (green); unassigned residues (gray); peptide from pMHC (pink); Cβ FG loop (yellow).

Figure 8:

Molecular dynamics simulations of pMHC binding to MHC class I- and class II-restricted TCR ectodomains. (A) ΔRMSF (unbound TCR – bound TCR) values for the MHC class II-restricted MS2–3C8 β chain. MS2–3C8 β chain residues that become more rigid upon binding to MBP-HLA-DR4 are positive, while residues with a gain in flexibility are negative. Residue positions of the CDR loops and the FG loop are highlighted. Error bars indicate ±1SD. (B) ΔRMSF values for the MHC class I-restricted TCR A6 β chain [19]. (C) Mapping of ΔRMSF values onto the β chain of MS2–3C8 in complex with MBP–HLA-DR4 (PDB 3O6F) [23]. Color coding: HLA-DR4 (gray); MBP peptide (pink); MS2–3C8 α chain (green); MS2–3C8 β chain (wheat); ΔRMSF > 0.2 Å (blue); 0.1 < ΔRMSF < 0.2 Å (light blue); −0.2 < ΔRMSF < −0.1 Å (orange); and ΔRMSF < −0.2 Å (red). (D) Mapping of ΔRMSF values onto the β chain of TCR A6 in complex with Tax–HLA-A2 (PDB 1QRN) [38]. Color coding: HLA-A2 (gray); Tax peptide (pink); A6 α chain (green); A6 β chain (wheat). ΔRMSF values are color coded as in (C).