Structure of Staphylococcal Enterotoxin E in Complex with TCR Defines the Role of TCR Loop Positioning in Superantigen Recognition

<div><p>T cells are crucial players in cell-mediated immunity. The specificity of their receptor, the T cell receptor (TCR), is central for the immune system to distinguish foreign from host antigens. Superantigens are bacterial toxins capable of inducing a toxic immune response by cross-linking the TCR and the major histocompatibility complex (MHC) class II and circumventing the antigen specificity. Here, we present the structure of staphylococcal enterotoxin E (SEE) in complex with a human T cell receptor, as well as the unligated T cell receptor structure. There are clear structural changes in the TCR loops upon superantigen binding. In particular, the HV4 loop moves to circumvent steric clashes upon complex formation. In addition, a predicted ternary model of SEE in complex with both TCR and MHC class II displays intermolecular contacts between the TCR α-chain and the MHC, suggesting that the TCR α-chain is of importance for complex formation.</p></div

Comparison between structurally determined CDR2 loops.

<p>(A) Close-up view of the CDR2β loop of 23 structurally determined TCRs. TRBV domains with coloured loops have been reported to bind SEE, while the non-binders are shown in grey; TRBV4-1 (pink), TRBV5-1 (orange), TRBV7-2 (green), TRBV7-3 (purple), TRBV7-8 (cyan), TRBV7-9 (blue), TRBV11-2 (brown), TRBV12-4 (red), and TRBV14 (yellow), (B) comparison between the SEE-TRBV7-9 structure in beige and blue, respectively, and TRBV19 in grey, which do not bind SEE. The hydrogen bond pattern to the backbone of CDR2 and the C” strand is shown with dotted lines.</p

X-ray structure of the SEE-TCR complex.

<p>(A) Overall structure of the complex, with SEE in beige, the TCR α-chain in purple and the β-chain in blue. (B) Close-up of the SEE α₂-helix and contacting residues in TCR, (C) the hydrophobic patch, (D) the α₄-β₉ loop, and (E) the upper part of the α₅-helix. Hydrogen bonds are marked as dotted lines.</p

Data collection and refinement statistics.

<p>Data were collected from a single crystal. Values in parentheses are for the highest resolution shell.</p><p>Data collection and refinement statistics.</p

Modelling of the TCR-SEE-(MHC)₂ quaternary complex.

<p>(A) Sequence alignment of SEE with SEA, SEB, SEH and SEI, displaying the conservation of both MHC binding sites in SEE, made using ClustalW2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131988#pone.0131988.ref084" target="_blank">84</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131988#pone.0131988.ref085" target="_blank">85</a>]. The N-terminal binding site to the MHC α-chain is marked in green and the C-terminal binding site to the MHC β-chain is marked in purple. (B) The initial model of TCR-SEE-(MHC)₂. The TCR is shown in purple and blue (TCRα and TCRβ respectively), the SEE in beige, and MHC molecules in green. (C) The final model of TCR-SEE-(MHC)₂. (D) The TCR-SEB-MHC structure, with SEB shown in orange.</p

Purification of natively folded SEA, SEE and SEH.

<p>(A, C, E) Size-exclusion chromatography of SEA, SEE and SEH, with protein purities analyzed by coomassie-stained SDS-PAGE gels shown as insets. (B, D, F) Far-UV CD spectra for SEA, SEE, SEH at 200–250 nm at 20°C in presence of 0.1 mM ZnCl₂ (dark grey) or 1 mM EDTA (light grey) at pH 6.0.</p

Evaluation of secondary structure for SEA and SEE after heating and re-cooling.

<p>Transition curves upon heating (40°C—95°C, dark grey) and cooling (40°C, light grey) were recorded by CD spectroscopy at 220 nm for (A) SEA and (D) SEE at pH 5.0 in presence of 0.1 mM ZnCl₂. Far-UV CD spectra (195–250 nm) for (B) SEA and (E) SEE at pH 5.0 in presence of 0.1 mM ZnCl₂ recorded at 40°C (blue), 95°C (red), and after subsequent cooling to 40°C (dark blue). Quantification of secondary structural elements using the far-UV CD spectra for (C) SEA and (F) SEE at 40°C (blue), upon heating to 95°C (red) and cooling back to 40°C (dark blue). The RMSD between experimental and fitted data for SEA is: 0.235, 0.615 and 0.152 for initial 40°C, 95°C and after subsequent cooling to 40°C, respectively. The RMSD values for SEE are 0.157, 0.179 and 0.153, respectively, for the same conditions.</p

Transition temperatures (T_s values, [°C]) of SE secondary structures at pH 5.0–7.0 in presence of EDTA or Zn²⁺.

<p>Transition temperatures (T_s values, [°C]) of SE secondary structures at pH 5.0–7.0 in presence of EDTA or Zn²⁺.</p

Intrinsic fluorescence for SEA, SEE and SEH as a function of temperature.

<p>Thermal tertiary structure changes of SEA, SEE and SEH were monitored by tryptophan/tyrosine fluorescence. (A) SEA in the presence of 0.1 mM ZnCl₂ at pH 5.0, (B) SEE in the presence of 0.1 mM ZnCl₂ at pH 5.0, (C) SEH at pH 7.0 in the presence of 0.1 mM ZnCl₂ or (D) SEH at pH 7.0 in the presence of 1 mM EDTA. The excitation wavelength was 280 nm. Temperatures are represented by colors from blue, 20°C to red, 90°C. Black arrows indicate fluorescence emission maxima red- or blue-shifting.</p

Thermal denaturation of SEA, SEE and SEH at different pH values.

<p>Transition curves upon heating for (A) SEA, (B) SEE and (C) SEH in presence of 0.1 mM ZnCl₂ (dark grey) or 1 mM EDTA (light grey) measured by CD spectroscopy at 220 nm, from 40–95°C.</p