A Mutational Analysis of the Binding of Staphylococcal Enterotoxins B and C3 to the T Cell Receptor β Chain and Major Histocompatibility Complex Class II

Abstract

The three-dimensional structure of the complex between a T cell receptor (TCR) β chain (mouse Vβ8.2Jβ2.1Cβ1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 Å resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the β–SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR β chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR β chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR–SAG and SAG–MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the β–SEC3 complex (“hot spot” residues) are strictly conserved among enterotoxins reactive with mouse Vβ8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vβ-binding specificities.

Immune regulatory loops determine productive interactions within human T lymphocyte–dendritic cell clusters

“…Expression of CD70 and CD27 antigens was monitored over a period of 6 days. SAG were used to provide a means of polyclonal T cell activation under conditions mimicking best T cell antigen receptor͞MHC͞peptide affinities in the presence of DC costimulation (23,24). Consistent with previously published data, we found that CD8 ϩ CTL uniformly and rapidly up-regulate CD70 molecules, with peak expression between day 2 and 4 as assessed by three-color flow cytometry analysis ( Fig.…”

Functional Analysis of the TCR Binding Domain of Toxic Shock Syndrome Toxin-1 Predicts Further Diversity in MHC Class II/Superantigen/TCR Ternary Complexes

“…Thus, it appears that the high affinity MHC binding site can circumvent the requirement for stabilizing interactions between the TCR and MHC class II (19). Mutagenesis of SEC3 showed a direct correlation between the affinity of SEC3 for the TCR ␤-chain and mitogenicity (5). Recently, phage display was used to engineer SEC3 variants in the disulfide loop with increased affinities to the ␣/␤ TCR revealing that increasing affinity of the SEC3/TCR complex above that normally seen with SAG/TCR interactions resulted in increased T cell activation (8).…”

“…Andersen et al (6) have elegantly shown that the TCR ␣-chain interacts with the MHC ␤-chain to stabilize the trimeric complex involving SEB. Although, the TCR/SPE C/MHC class II model predicts that in this interaction SAG acts as a bridge without any direct interactions between TCR and MHC class II, SPE C contains a high affinity (K d ϭ ϳ 4 ϫ 10 Ϫ8 M), zinc-dependent, MHC class II binding domain (19), and small changes in MHC affinity can overcome larger increases in TCR affinity (5). Thus, it appears that the high affinity MHC binding site can circumvent the requirement for stabilizing interactions between the TCR and MHC class II (19).…”

“…Based on the cocrystal structure of SEC3 in contact with the mouse 14.3.d ␤-chain (21), the mutation of all SEC3 residues involved in contact with the ␤-chain revealed that only two of 12 residues (N23A, Q210A) completely lost detectable binding using their criteria for stimulatory capacity, while a third mutation (F176A) had barely detectable mitogenic activity (5). Most other SEC3 mutations did show more intermediate decreases in stimulatory capacity, which we did not observe for the TSST-1 mutants, although three SEC3 mutants showed similar minor decreases in mitogenic activity (5). These residues (Gly 102 , Lys 103 , and Gly 106 in SEC3) are located on the flexible disulfide loop and do not form hydrogen bonds with the 14.3.d TCR (21).…”

Systems Biology Approach to Identifying Host Interactive Pathways Modulating the Severity of Streptococcal Sepsis

“…While this association made perfect biological sense based on the known central role of SAgs in the human disease, mice are not susceptible to SAgs due to an inherent lower affinity of mouse MHC class II molecules to GAS SAgs. The role of GAS SAgs can be well investigated in HLA class II transgenic mice as others and we have reported (Leder et al, 1998;Andersen et al, 1999;Welcher et al, 2002;Nooh et al, 2007). However, due to the overwhelming response to SAgs in these mice, it is difficult to use them to dissect host response to other GAS virulence factors in the disease process.…”