The T cell receptor beta-chain second complementarity determining region loop (CDR2beta governs T cell activation and Vbeta specificity by bacterial superantigens

Abstract

Superantigens (SAgs) are microbial toxins defined by their ability to activate T lymphocytes in a T cell receptor (TCR) β-chain variable domain (Vβ)-specific manner. Although existing structural information indicates that diverse bacterial SAgs all uniformly engage the Vβ second complementarity determining region (CDR2β) loop, the molecular rules that dictate SAg-mediated T cell activation and Vβ specificity are not fully understood. Herein we report the crystal structure of human Vβ2.1 (hVβ2.1) in complex with the toxic shock syndrome toxin-1 (TSST-1) SAg, and mutagenesis of hVβ2.1 indicates that the non-canonical length of CDR2β is a critical determinant for recognition by TSST-1 as well as the distantly related SAg streptococcal pyrogenic exotoxin C. Frame work (FR) region 3 is uniquely critical for TSST-1 function explaining the fine Vβ-specificity exhibited by this SAg. Furthermore, domain swapping experiments with SAgs, which use distinct domains to engage both CDR2β and FR3/4β revealed that the CDR2β contacts dictate T lymphocyte Vβ-specificity. These findings demonstrate that the TCR CDR2β loop is the critical determinant for functional recognition and Vβ-specificity by diverse bacterial SAgs.

FIGURE 1.

Phylogenetic relationships and representative Vβ-binding topologies for the bacterial SAgs. A, phylogenetic tree of known bacterial SAgs. The unrooted tree was based on the alignment of amino acid sequences using Clustal W (47) and constructed with the unweighted pair group method using arithmetic averages (UPGMA) in MacVector 7.2.3. The SAg abbreviations are indicated followed by the relevant accession number. As previously proposed (7), the five main groups of SAgs belonging to the pyrogenic toxin class are indicated. MAM, YPM, and non-Group A streptococcal SAgs are also included in the analysis. The number of times each branch was supported from 1000 bootstraps is shown as a percentage. B, overview of SAg engagement of the T cell receptor Vβ domain. Ribbon diagrams (left panel) of the TSST-1-Vβ2.1, SEB-Vβ8.2 (19), SpeC-Vβ2.1 (21), SEK-Vβ5 (18), and MAM-Vβ8.2/Vα3 (22) complexes. The Vβ domains (in gray) are aligned to one another to highlight the distinct regions of the apical side of the Vβ domains engaged by these SAgs, and the mouse Vα3 (mVα3) (in orange) from the MAM-Vβ8.2/Vα3 structure is modeled onto the other structures for comparison. Surface-filled models (right panel) of the corresponding Vβ domains with the molecular surface buried by the corresponding SAgs are demarcated. CDR, HV, and FR regions buried in the interface by the corresponding SAg are color-coded as indicated in the legend.

FIGURE 2.

Exposition of the differences between conventional TCR-pMHC and TCR-SAg-pMHC T cell signaling complexes. A, cartoon representation of conventional TCR-pMHC ternary complex (36), where the TCR adopts a semi-conserved diagonal docking mode on pMHC II. By and large, the CDR1 and CDR2 loops engage the α-helices of MHC II and the CDR3 loops engage the antigenic peptide. B, cartoon representation of the TSST-1-mediated T cell activation complex (20), where TSST-1 acts a bridge between TCR and pMHC II complex and direct TCR contact with pMHC II is abrogated, thus removing antigenic peptide recognition. C, cartoon representation of the SpeC-mediated T cell activation complex (11, 16, 21) where SpeC cross-links MHC II molecules through zinc-mediated, high-affinity binding to the pMHC II β-chain (left) (16), and as a wedge, by binding to the low-affinity binding site on the pMHC II α-chain (11) and preventing direct contacts between TCR Vβ and the antigenic peptide. Colors are as follows: MHC α-chain, green; MHC β-chain, red; antigenic peptide, black; TCR α-chain, orange; TCR β-chain, gray; Zn²⁺ ion, magenta; TSST-1, yellow and SpeC, blue. D, close up view of hVβ2.1 (PDB code 3MFG) showing all the residues involved in binding to TSST-1. E, close up view of hVβ2.1 (PDB code 1KTK) showing all residues involved in binding to SpeC.

FIGURE 3.

Functional analysis of TSST-1 and SpeC binding interfaces on the hVβ2.1 TCR. A, titration of TSST-1 and SpeC to optimize IL-2 secretion from HuT78-hVβ2.1 T cells. IL-2 secreted from electroporated HuT78 T cells transiently expressing hVβ2.1 mutations within CDR1 (B), CDR2 (C), Ser^52a (D), and FR3 (E), and CDR3 and HV4 (F). For panels (B–F), electroporated HuT78 T cells expressing eGFP and wild-type hVβ2.1 were used as negative and positive controls, respectively, with SpeC and TSST-1 added at a final concentration of 1 μg/ml, and the internal control anti-Vβ2/anti-CD28 beads added at one bead per electroporated HuT78 T cells. Data shown are the average ± S.E. from at least three independent experiments each conducted in triplicate (*, p < 0.05 compared with HuT78-Vβ2.1, and **, p < 0.05 compared with HuT78 transfected with wild-type hVβ2.1 but not significantly different compared with HuT78 transfected with the eGFP negative control).

FIGURE 4.

Functional hotspots of TSST-1 and SpeC engage the CDR2β loop of hVβ2.1. Top, TSST-1-hVβ2.1 complex. Bottom, SpeC-hVβ2.1 complex. The middle panels show ribbon diagrams of each SAg-Vβ complex, and the left and right panels show space-filling models for hVβ2.1, and TSST-1 or SpeC, respectively, rotated away from the central panel around the y axis as indicated. SAg residues critical for function are colored magenta. hVβ2.1 residues critical for function are colored red, and those with moderate phenotypes are colored yellow.

FIGURE 5.

Group V SAgs interact with their Vβ ligand through two distinct domains. A, cartoon representations of two homologous Group V SAgs interacting with their Vβ ligand. Both SEK and SpeI interact with the CDR2β loop with their Nα domain, and with the FR3/4β regions with α3-β8 loop. Colors are as follows: Vβ domain, gray; SEK, blue; SpeI, red; SEK Nα domain, green; SEK α3-β8 loop, magenta; SpeI Nα domain, yellow, and SpeI α3-β8 loop, cyan. B, amino acid sequence alignment of SEK and SpeI showing the Nα domain and α3-β8 loop sequences used in the domain-swapping mutants. C, graphic representations of the domain swapping mutants colored coded as in panel A. D, SDS-PAGE of the purified the wild-type SpeI and SEK or hybrid proteins.

FIGURE 6.

The N-terminal α-helix (Nα) domain of Group V SAgs governs Vβ specificity through contacts with CDR2β. qRT-PCR analysis of Vβ expression from human PBMC stimulated with 1 μg/ml of the wild-type SpeI and SEK or hybrid proteins shown in Fig. 5D. The data were normalized and set at 1.0 (dashed line) to responses from the wild-type SAg for the corresponding Vβ target (SpeI targets hVβ6.7, 9 and 21.3 while SEK targets hVβ5). Data are the average ± S.E. from three independent donors each done in triplicate (n = 9). Statistical significance was determined between the values from wild-type SAg and corresponding domain-swapped mutants, and values between the Nα domain-swapped mutants and the double Nα/α3β8 loop-swapped mutants (*, p < 0.05; **, p < 0.01; and ***, p < 0.001).