The TCR forms a complex with CD3 proteins

Overview (A) of the TCR and the CD3 proteins. A superscript is used to distinguish the CD3ε and CD3ζ chains. CD3εδ refers to the CD3ε from the CD3δε dimer and likewise CD3εγ refers to the CD3ε of the CD3γε dimer. CD3ζγ refers to the CD3ζ closest to the transmembrane (TM) region of CD3γ and CD3ζε to the CD3ζ closest to the TM region of CD3εδ. Structure from PDB code 6JXR15. Snapshots of the TCR-CD3 (B) and TCR-CD3-pMHC (C) simulation systems. The TCR (green), CD3 proteins (cyan), pMHC (purple), and lipid headgroups (light brown) are shown. In the TCR-CD3-pMHC system a pore is present in the upper membrane to allow the flow of solvent between the two compartments, which is why headgroup beads are visible in the membrane interior. Solvent and lipid tails are not shown for clarity. In (C), the TCR variable and constant regions are indicated, as well as the ITAMs on the CD3 proteins. The TCR extracellular domain (EC) encompasses the variable and constant regions.

Distribution of CD3ε chains around TCR αβ visualized by iso-occupancies and polar plots

Iso-occupancy of CD3εγεδ in the TCR-CD3 simulation (A) and in the TCR-CD3-pMHC simulation (B). The TCR (lime), CD3 (cyan), which are mostly obscured by the iso-surfaces, and pMHC (purple) are shown. Polar plots of CD3εγ chains around TCRβ chain in the TCR-CD3 simulation (C) and in the TCR-CD3-pMHC simulation (D).

TCR-CD3 contacts

The number of contacts between the TCRβ variable domain and the indicated CD3 chains in the TCR-CD3 (green) and TCR-CD3-pMHC (purple) simulations. For clarity outliers are not shown. Contacts per residue are shown in Figure S2.

The TCR adopts a different conformation in the TCR-CD3 and TCR-CD3-pMHC simulations

(A) Illustration of the atoms used for the tilt angle calculation, which were TCRβ asparagine 97 TCRβ serine 249 and TCRβ valine 281. (B) Distribution of the tilt of the TCR EC (TCRβ EC-TCRβ TM angle) in the TCR-CD3 (green) and TCR-CD3-pMHC (purple) systems. The TCR EC tilt in the published cryo-EM structure15 (vertical line) was 144°. (C) Snapshots of the TCR-CD3 (left) and TCR-CD3-pMHC (right) simulations. Note the difference in the tilt angle. The TCR (green), CD3 proteins (cyan) and part of the pMHC (purple) are shown.

TCR-pMHC binding acts as a drawbridge for CD3 movement

An unbound TCR clamps the CD3 proteins, which prevents CD3 diffusion around the TCR in the membrane (left). Upon pMHC binding (right), the TCR becomes extended, which weakens binding to CD3 and allows CD3 diffusion around the TCR. Increased CD3 movement in the membrane facilitates the release of CD3 tails in the cytosol, where the ITAMS are accessible for phosphorylation by Lck.

Contacts between TCRɑ AB loop, TCRβ H3 helix and the TCRβ FG loop and the CD3 proteins

(A) Side (left) and top view (right) of the TCR-CD3 cryo-EM structure. The TCRɑ AB loop, TCRβ H3 helix and the TCRβ FG loop are located far from the TCR-pMHC binding interface and are out of reach of the CD3 proteins. The CD3 proteins with extracellular domains (CD3δ, CD3ε and CD3γ) are located at the opposite side of the TCRɑβ chains, while the CD3ζ chains lack an extracellular domain. Approximate TCR-pMHC binding face is indicated. TCRɑ in green with the AB loop in fuchsia, TCRβ is shown in yellow with the H3 helix in purple and the FG loop in blue, the CD3 protein chains are colored grey for clarity. Structure from PDB code 6JXR15. Cumulative contacts between different CD3 chains and the TCRα AB loop (B), TCRβ H3 helix (C), and the TCRβ FG loop (D) in the TCR-CD3 (green) and TCR-CD3-pMHC simulations (purple). Contacts are summed over all simulations and shown as a graded color, where each step in the gradient represents contacts from a single simulation. Residue numbers in the TCRα or TCRβ chains are shown on the x-axis. (E-F) Simulation snapshots illustrating the role of the TCRβ FG loop. In the TCR-CD3 simulations the FG loop blocked the diffusion of CD3εγ (panel E). Upon TCR-pMHC binding, the TCR elongated, and the FG loop moved up, allowing the diffusion of the CD3 proteins alongside TCRβ. TCRβ is shown in yellow with the FG loop in blue, CD3εγ in viridian green, and the pMHC in purple, other protein chains are colored grey for clarity.

Hinge rigidification results in TCRs that are unable to retain the CD3 subunits

(A) Multiple sequence alignment of the TCRβ connecting peptide (CP) with the two conserved glycines in pink. (B) CP region of the TCR. The TCRɑ CP is longer than the TCRβ CP, but the TCRβ CP contains two flexible glycines. TCRɑ (green) with CP (blue), TCRβ (yellow) with CP (pink), and CP glycines (spheres) are shown. Structure from PDB code 6JXR15. (C) Atomistic molecular dynamics simulations for wild-type GG and “rigidified” (PP and AA) TCR mutants indicated that movement of CD3εγ (represented as densities in a 2D plane about the TCR) increased in the PP simulations compared to that in wild type.

Hinge rigidification results in hyperresponsive T cells

NFAT reporter cells were transfected with GG, AA, or PP mutant TCRs. Cells with the PP TCRs, but not GG or AA TCRs, were hyperresponsive to pMHC stimulation after 2 h, but the difference between mutant and wild type cells decreased after 4 h and 24 h. Flow cytometry dot plots (A) and histogram (B) identifying GFP-positive T cells after co-culturing with APC and 1 µg/ml HEL peptide for 2 h. Results of a representative experiment are shown. (C) Percentage of GFP expressing T cells after 2, 4 or 24 h of co-culture. Note that the y-axis varies between the plots. Data are represented as mean ± stdev for all replicates from 2-3 independent experiments. (D) Percentage of GFP-positive 3A9 T cells after being stimulated with 1 μg/ml HEL peptide over different time points (2, 4, 24 h) Data are represented as mean ± stdev for 3 replicates from one representative experiment. n.s.: non-significance, ****: P <0.0001 for PP compared to wild type (GG), **: P <0.01 for PP compared to wild type (GG).

Residues included in the simulation. In the ITAM column, the numbers between the brackets refer to the uniprot numbering #The CD3ε ITAM sequence length differs between mouse and human, the numbers refer to mouse sequence. *Residues 181-189 of the CD3ε chains (TCR E and TCR F) were omitted during modeling and not included in the simulation. $Numbering scheme of 3qiu is used, chain C for TCR ⍺ and chain D for TCRβ.

Residues included in the simulation. In the ITAM column, the numbers between the brackets refer to the uniprot numbering #The CD3ε ITAM sequence length differs between mouse and human; the numbers refer to the mouse sequence. *Residues 181-189 of the CD3εδ and CD3εγ chains (TCR E and TCR F) were omitted during modeling and not included in the simulation. $Numbering scheme of 3qiu is used, chain C for TCR⍺ and chain D for TCRβ.

TCR and MHC residues used for reconstruction. Unless indicated, residues were reconstructed using Spanner52.

Harmonic potentials used to restrain the epitope in the MHC.

Disulfide bonds present in the simulation. +Interchain disulfide bond are modeled by a harmonic potential.

List of distance restraints between the TCR and the pMHC. During the docking simulations, the force constant was set to 1 kJ mol−1 nm−2. A piecewise linear/harmonic restraint was used (bond type 10 in Gromacs) to keep the TCR and the pMHC together. The potential is quadratic for rij < r0, 0 for r0 ≤ rij < r1, quadratic for r1 ≤ rij < r2, and linear for r2 ≤ rij.