The Celiac-Disease Superantigen Oligomerizes and Increases Permeability in an Enterocyte Cell Model.

Celiac disease (CeD) is an autoimmune disorder triggered by gluten proteins, affecting approximately 1 % of the global population. The 33-mer deamidated gliadin peptide (DGP) is a metabolically modified wheat-gluten superantigen for CeD. Here, we demonstrate that the 33-mer DGP spontaneously assembles into oligomers with a diameter of approximately 24 nm. The 33-mer DGP oligomers present two main secondary structural motifs-a major polyproline II helix and a minor ß-sheet structure. Importantly, in the presence of 33-mer DGP oligomers, there is a statistically significant increase in the permeability in the gut epithelial cell model Caco-2, accompanied by the redistribution of zonula occludens-1, a master tight junction protein. These findings provide novel molecular and supramolecular insights into the impact of 33-mer DGP in CeD and highlight the relevance of gliadin peptide oligomerization.

Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level.

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.

Heterologous Chimeric Construct Comprising a Modified Bacterial Superantigen and a Cruzipain Domain Confers Protection Against Trypanosoma cruzi Infection.

Chagas disease is an endemic chronic parasitosis in Latin America affecting more than 7 million people. Around 100 million people are currently at risk of acquiring the infection; however, no effective vaccine has been developed yet. Trypanosoma cruzi is the etiological agent of this parasitosis and as an intracellular protozoan it can reside within different tissues, mainly muscle cells, evading host immunity and allowing progression towards the chronic stage of the disease. Considering this intracellular parasitism triggers strong cellular immunity that, besides being necessary to limit infection, is not sufficient to eradicate the parasite from tissues, a differential immune response is required and new strategies for vaccines against Chagas disease need to be explored. In this work, we designed, cloned and expressed a chimeric molecule, named NCz-SEGN24A, comprising a parasite antigen, the N-terminal domain of the major cysteine protease of T. cruzi, cruzipain (Nt-Cz), and a non-toxic form of the staphylococcal superantigen (SAg) G, SEG, with the residue Asn24 mutated to Ala (N24A). The mutant SAg SEGN24A, retains its ability to trigger classical activation of macrophages without inducing T cell apoptosis. To evaluate, as a proof of concept, the immunogenicity and efficacy of the chimeric immunogen vs. its individual antigens, C3H mice were immunized intramuscularly with NCz-SEGN24A co-adjuvanted with CpG-ODN, or the recombinant proteins Nt-Cz plus SEGN24A with the same adjuvant. Vaccinated mice significantly produced Nt-Cz-specific IgG titers after immunization and developed higher IgG2a than IgG1 titers. Specific cell-mediated immunity was assessed by in-vivo DTH and significant responses were obtained. To assess protection, mice were challenged with trypomastigotes of T. cruzi. Both schemes reduced the parasite load throughout the acute phase, but only mice immunized with NCz-SEGN24A showed significant differences against control; moreover, these mice maintained 100% survival. These results encourage testing mutated superantigens fused to specific antigens as immune modulators against pathogens.

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy.

BACKGROUND: Variable heavy chain (VH) family frameworks (FWRs) have been reported to affect antibody receptor and superantigen binding; however, such effects in IgE remain largely unknown. Given that VH family biases have been previously reported in IgE of certain allergies, there is a need to investigate this phenomenon for biotechnological and therapeutic purposes. OBJECTIVE: We sought to investigate the effects of VH families on IgE interaction with FcεRIα, anti-IgE omalizumab, antigen, and superantigen protein A (spA) by using the pertuzumab and trastuzumab IgE models. METHODS: Pertuzumab VH1-VH7 family variants of IgE with the same complementarity-determining regions were investigated with regard to their binding interactions to FcεRIα, Her2, omalizumab, and spA. Notable FcεRIα-IgE observations were cross-checked against appropriate trastuzumab IgE VH variants. Computational structural modeling and simulations were also performed for insight into the mechanism of interactions with various VH FWRs. RESULTS: The pertuzumab VH5 IgE variant, but not the trastuzumab VH5 IgE, was found to interact with FcεRIα significantly longer than the respective VH family variants within each model antibody. No significant differences in interaction were found between IgE and omalizumab for the pertuzumab VH variants. Although trastuzumab VH3 interacted with spA, none of our pertuzumab VH variants, including VH3, associated with spA. CONCLUSION: We found unexpected varying allosteric communications caused by the VH family FWRs to the FcεRIα-, Her2-, and spA-binding regions of pertuzumab IgE, with implications for use of IgE/anti-IgE therapeutics to treat allergy and IgE therapeutics in allergo-oncology.

Mutations to Cysteine Residues in the Trypanosoma cruzi B-Cell Superantigen Tc24 Diminish Susceptibility to IgM-Mediated Hydrolysis.

B-cell superantigens (BC-SAgs) are immunoevasins that have evolved in response to innate catalytic IgM antibodies; germ-line encoded immunoglobulins present in the preimmune repertoire independent of prior antigen exposure. Catalysis is the result of a 2-step process that involves first the formation of a non-covalent bond between the BC-SAg and the immunoglobulin followed by covalent bond formation at the catalytic site resulting in target hydrolysis. Tc24 is a recently described Trypanosoma cruzi BC-SAg hypothesized to play a role in evading the humoral response early in the infection period. We previously demonstrated that exposure to Tc24 following immunization or infection resulted in the depletion of the catalytic IgM response, leaving a gap in the catalytic IgM repertoire. The present report compares the BC-SAg properties of wild-type Tc24 (Tc24-WT) to that of 2 recombinant Tc24 isoforms: Tc24-C2 (Cys to Ser mutations in the 2 most-proximal Cys residues) and Tc24-C4 (Cys to Ser mutations in all 4 Cys residues present). BC-SAg activity was assessed by immunizing mice with the respective isoforms and examining the ability of IgM purified from the respective groups to hydrolyze the 3 Tc24 isoforms. In addition, the ability of IgM purified from naive mice to hydrolyze the Tc24 isoforms was also assessed. Immunization with Tc24-WT, Tc24-C2, or Tc24-C4 resulted in loss of IgM-mediated hydrolysis of Tc24-WT. However, the ability of IgM purified from naive mice (previously shown to hydrolyze Tc24-WT) was less effective in hydrolyzing the 2 Tc24 isoforms. These data demonstrate that although the BC-SAg site in the mutants remained intact, their reduced susceptibility to IgM-mediated hydrolysis suggested that structural changes resulting from the Cys to Ser mutations altered accessibility to the catalytic site in the 2 isoforms.

Superantigens hyperinduce inflammatory cytokines by enhancing the B7-2/CD28 costimulatory receptor interaction.

Full T-cell activation requires interaction between the costimulatory receptors B7-2 and CD28. By binding CD28, bacterial superantigens elicit harmful inflammatory cytokine overexpression through an unknown mechanism. We show that, by engaging not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the avidity between B7-2 and CD28, inducing thereby T-cell hyperactivation. Using the same 12-aa ß-strand-hinge-α-helix domain, superantigens engage both B7-2 and CD28 at their homodimer interfaces, areas remote from where these coreceptors interact, implying that inflammatory signaling can be controlled through the receptor homodimer interfaces. Short B7-2 dimer interface mimetic peptides bind diverse superantigens, prevent superantigen binding to cell-surface B7-2 or CD28, attenuate inflammatory cytokine overexpression, and protect mice from lethal superantigen challenge. Thus, superantigens induce a cytokine storm not only by mediating the interaction between MHC-II molecule and T-cell receptor but also, critically, by promoting B7-2/CD28 coreceptor engagement, forcing the principal costimulatory axis to signal excessively. Our results reveal a role for B7-2 as obligatory receptor for superantigens. B7-2 homodimer interface mimotopes prevent superantigen lethality by blocking the superantigen-host costimulatory receptor interaction.

Pfit is a structurally novel Crohn's disease-associated superantigen.

T cell responses to enteric bacteria are important in inflammatory bowel disease. I2, encoded by the pfiT gene of Pseudomonas fluorescens, is a T-cell superantigen associated with human Crohn's disease. Here we report the crystal structure of pfiT at 1.7Å resolution and provide a functional analysis of the interaction of pfiT and its homolog, PA2885, with human class II MHC. Both pfiT and PA2885 bound to mammalian cells and stimulated the proliferation of human lymphocytes. This binding was greatly inhibited by anti-class II MHC HLA-DR antibodies, and to a lesser extent, by anti HLA-DQ and DP antibodies, indicating that the binding was class II MHC-specific. GST-pfiT efficiently precipitated both endogenous and in vitro purified recombinant HLA-DR1 molecules, indicating that pfiT directly interacted with HLA-DR1. Competition studies revealed that pfiT and the superantigen Mycoplasma arthritidis mitogen (MAM) competed for binding to HLA-DR, indicating that their binding sites overlap. Structural analyses established that pfiT belongs to the TetR-family of DNA-binding transcription regulators. The distinct structure of pfiT indicates that it represents a new family of T cell superantigens.

Gram-positive bacterial superantigen outside-in signaling causes toxic shock syndrome.

Staphylococcus aureus and Streptococcus pyogenes (group A streptococci) are Gram-positive pathogens capable of producing a variety of bacterial exotoxins known as superantigens. Superantigens interact with antigen-presenting cells (APCs) and T cells to induce T cell proliferation and massive cytokine production, which leads to fever, rash, capillary leak and subsequent hypotension, the major symptoms of toxic shock syndrome. Both S. aureus and group A streptococci colonize mucosal surfaces, including the anterior nares and vagina for S. aureus, and the oropharynx and less commonly the vagina for group A streptococci. However, due to their abilities to secrete a variety of virulence factors, the organisms can also cause illnesses from the mucosa. This review provides an updated discussion of the biochemical and structural features of one group of secreted virulence factors, the staphylococcal and group A streptococcal superantigens, and their abilities to cause toxic shock syndrome from a mucosal surface. The main focus of this review, however, is the abilities of superantigens to induce cytokines and chemokines from epithelial cells, which has been linked to a dodecapeptide region that is relatively conserved among all superantigens and is distinct from the binding sites required for interactions with APCs and T cells. This phenomenon, termed outside-in signaling, acts to recruit adaptive immune cells to the submucosa, where the superantigens can then interact with those cells to initiate the final cytokine cascades that lead to toxic shock syndrome.

Solid tumor-targeted infiltrating cytotoxic T lymphocytes retained by a superantigen fusion protein.

Successful immune-mediated regression of solid tumors is difficult because of the small number of cytotoxic T lymphocytes (CTLs) that were traffic to the tumor site. Here, the targeting of tumor-specific infiltrating CTLs was dependent on a fusion protein consisting of human epidermal growth factor (EGF) and staphylococcal enterotoxin A (SEA) with the D227A mutation. EGF-SEA strongly restrained the growth of murine solid sarcoma 180 (S180) tumors (control versus EGF-SEA, mean tumor weight: 1.013 versus 0.197 g, difference  = 0.816 g). In mice treated with EGF-SEA, CD4+, CD8+ and SEA-reactive T lymphocytes were enriched around the EGFR expressing tumor cells. The EGF receptors were potentially phosphorylated by EGF-SEA stimulation and the fusion protein promoted T cells to release the tumoricidal cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Intratumoral CTLs secreted cytolytic pore-forming perforins and granzyme B proteins near the surface of carcinomas, causing the death of many tumor cells. We additionally show that labeled EGF-SEA was directly targeted to the tumor tissue after intravenous (i.v.) injection. The findings demonstrate that antibody-like EGF-SEA plays an important role in arresting CTLs in the solid tumor site and has therapeutic potential as a tumor-targeting agent.

Preparation and in-vitro bioactivity of a novel superantigen conjugate targeting bladder carcinoma.

OBJECTIVES: Superantigens have shown potent effects against bladder tumours by inducing Vbeta-specific T-lymphocyte proliferation and massive cytokine release but therapeutic benefit is compromised by cytotoxicity towards non-malignant cells and hypotoxicity to major histocompability complex (MHC) II-negative tumour cells. We are therefore interested in a conjugate preparation of a monoclonal antibody (MAb)-superantigens conjugate for which these drawbacks would be resolved. METHODS: The Fab fragment of the anti-bladder carcinoma MAb BDI-1 was conjugated to one member of the staphylococcal enterotoxin A (SEA) superantigen using the chemical conjugating reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. RESULTS: After HPLC purification through a Superdex-200 gel column, another peak with a molecular mass of 250 KDa was observed before Fab and SEA were eluted. Indirect immunocytochemical analysis and immunofluorescence tests showed that the cell membranes of most human bladder cancer cells were positively stained only by the conjugate, confirming the ability of the conjugate to target human bladder carcinoma. Peripheral blood mononuclear cell proliferation and cytokine release were similar with the conjugate and SEA. Cytotoxicity targeting in MHC II-negative bladder cancer cell lines, evaluated by flow cytometry, showed significant differences between the conjugate and SEA, whereas there was no difference in the Lovo colon cancer cell line. CONCLUSIONS: These findings indicate the conjugate of SEA protein and BDI-1 Fab fragment was prepared successfully and targeted bladder carcinoma in vitro.

Functional analysis of the disulphide loop mutant of staphylococcal enterotoxin C2.

The superantigen staphylococcal enterotoxin C2 (SEC2) tremendously activate T lymphocytes bearing certain T-cell receptor Vbeta domains when binding to MHC II molecules, which launches a powerful response of tumour inhibition in vitro as well as in vivo. However, the toxicity of SEC2 performed in clinic limited its broad application for immunotherapy. The previous studies suggested that the disulphide loop may be important for the toxicity of some SEs, which prompted us to investigate the potential roles of the disulphide loop in biological activity of SEC2. Site-directed mutagenesis was used to disturb the formation of the disulphide bond by substituting Ala or Ser for Cys-93 and Cys-110. The expressed mutants in Escherichia coli were used to determine their superantigen activity and toxicity. Results showed that all of the mutated proteins exhibited reduced abilities to induce T-cell proliferation and cytotoxic effects on tumour cells L929 and Hepa1-6, suggesting that the disulphide loop plays functional role in maintaining the maximal superantigen activity of SEC2. Furthermore, the toxicity assays in vivo showed that all of the mutants induced a reduced emetic and pyrogenic responses compared with native SEC2, which might be important for further construction of lowly toxic superantigen agent.

Novel toxic shock syndrome toxin-1 amino acids required for biological activity.

Superantigens interact with T lymphocytes and macrophages to cause T lymphocyte proliferation and overwhelming cytokine production, which lead to toxic shock syndrome. Staphylococcus aureus superantigen toxic shock syndrome toxin-1 is a major cause of menstrual toxic shock syndrome. In general, superantigen-secreting S. aureus remains localized at the vaginal surface, and the superantigen must therefore penetrate the vaginal mucosa to interact with underlying immune cells to cause toxic shock syndrome. A dodecapeptide region (toxic shock syndrome toxin-1 amino acids F119-D130), relatively conserved among superantigens, has been implicated in superantigen penetration of the epithelium. The purpose of this study was to determine amino acids within this dodecapeptide region that are required for interaction with vaginal epithelium. Alanine mutations were constructed in S. aureus toxic shock syndrome toxin-1 amino acids D120 to D130. All mutants maintained superantigenicity, and selected mutants were lethal when given intravenously to rabbits. Toxic shock syndrome toxin-1 induces interleukin-8 from immortalized human vaginal epithelial cells; however, three toxin mutants (S127A, T128A, and D130A) induced low levels of interleukin-8 compared to wild type toxin. When carboxy-terminal mutants (S127A to D130A) were administered vaginally to rabbits, D130A was nonlethal, while S127A and T128A demonstrated delayed lethality compared to wild type toxin. In a porcine ex vivo permeability model, mutant D130A penetrated the vaginal mucosa more quickly than wild type toxin. Toxic shock syndrome toxin-1 residue D130 may contribute to binding an epithelial receptor, which allows it to penetrate the vaginal mucosa, induce interleukin-8, and cause toxic shock syndrome.

A broad-spectrum inhibitory peptide against staphylococcal enterotoxin superantigen SEA, SEB and SEC.

The family of staphylococcal enterotoxin (SE) superantigen has been known to contribute to a broad-spectrum of diseases. In an attempt to screen the peptides with broad-spectrum inhibition against the superantigen activity of SEs, nine low-molecular peptides were designed on the basis of high conservative regions of amino acid sequences and structures of the SEs, the effects of these inhibitory peptides on the biological activity of SEs were detected by cellular and animal models, competition assay was used to detect the ability of the inhibitory peptide binding to MHC class II molecules. The results indicated that one dodecapeptide P72 possessed a broad-spectrum antagonist activity against superantigen SEA, SEB and SEC. The peptide P72 could protect mice from lethal toxic shock in the "two-hit" animal model. Competition experiment demonstrated peptide P72 could not bind to MHC class II molecules, which indicated the inhibitory activity of P72 may not due to binding to MHC class II. The peptide P72 with effective inhibition against the superantigen SEA, SEB and SEC may lead to a novel therapeutic modality in treatment of superantigen-mediated diseases.

Superantigen natural affinity maturation revealed by the crystal structure of staphylococcal enterotoxin G and its binding to T-cell receptor Vbeta8.2.

The illnesses associated with bacterial superantigens (SAgs) such as food poisoning and toxic shock syndrome, as well as the emerging threat of purpura fulminans and community-associated methicillin-resistant S. aureus producer of SAgs, emphasize the importance of a better characterization of SAg binding to their natural ligands, which would allow the development of drugs or biological reagents able to neutralize their action. SAgs are toxins that bind major histocompatibility complex class II molecules (MHC-II) and T-cell receptors (TCR), in a nonconventional manner, inducing T-cell activation that leads to production of cytokines such as tumor necrosis factor and interleukin-2, which may result in acute toxic shock. Previously, we cloned and expressed a new natural variant of staphylococcal enterotoxin G (SEG) and evaluated its ability to stimulate in vivo murine T-cell subpopulations. We found an early, strong, and widespread stimulation of mouse Vbeta8.2 T-cells when compared with other SAgs member of the SEB subfamily. In search for the reason of the strong mitogenic potency, we determined the SEG crystal structure by X-ray crystallography to 2.2 A resolution and analyzed SEG binding to mVbeta8.2 and MHC-II. Calorimetry and SPR analysis showed that SEG has an affinity for mVbeta8.2 40 to 100-fold higher than that reported for other members of SEB subfamily, and the highest reported for a wild type SAg-TCR couple. We also found that mutations introduced in mVbeta8.2 to produce a high affinity mutant for other members of the SEB subfamily do not greatly affect binding to SEG. Crystallographic analysis and docking into mVbeta8.2 in complex with SEB, SEC3, and SPEA showed that the deletions and substitution of key amino acids remodeled the putative surface of the mVbeta8.2 binding site without affecting the binding to MHC-II. This results in a SAg with improved binding to its natural ligands, which may confer a possible evolutionary advantage for bacterial strains expressing SEG.

Yersinia pseudotuberculosis superantigens.

Yersinia pseudotuberculosis, a gastro-intestinal bacterium, produces three closely related T cell superantigens, YPMa, YPMb and YPMc, which have no significant sequence similarity to other proteins, let alone other bacterial superantigens. Y. pseudotuberculosisderived mitogen (YPM) has been shown to play a role in the pathogenesis of human and animal Y. pseudotuberculosis infection. The three-dimensional structure of YPMa, as determined by X-ray crystallography and nuclear magnetic resonance spectroscopy, exhibits a jelly roll fold, a structural motif not observed in other superantigens. YPMa is structurally most similar to virus capsid proteins and members of the tumour necrosis factor (TNF) superfamily. In the crystal structure, YPMa forms a trimer, another feature shared with virus capsid proteins and TNF superfamily proteins. However, in solution YPMa exists as a monomer, and any functional relevance of the trimer observed in the crystals is yet to be established. Structures of YPM bound to the T cell receptor and/or the major histocompatibility complex (MHC) are not yet available and mapping of existing mutagenesis data onto the three-dimensional structure of YPMa did not reveal potential T cell receptor/MHC binding sites. Knowledge of the structure will aid the design of functional studies aimed at further characterizing this superantigen.

Crystal structure of a complete ternary complex of TCR, superantigen and peptide-MHC.

'Superantigens' (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-A resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR Vbeta but with TCR Valpha.

Immune response to MMTV infection.

Mouse mammary tumor virus (MMTV) has developed a strategy of exploitation of the immune response. It infects dendritic cells and B cells and requires this infection to establish an efficient chronic infection. This allows transmission of infection to the mammary gland, production in milk and infection of the next generation via lactation. The elaborate strategy developed by MMTV utilizes several key elements of the normal immune response. Starting with the infection and activation of dendritic cells and B cells leading to the expression of a viral superantigen followed by professional superantigen-mediated priming of naive polyclonal T cells by dendritic cells and induction of superantigen-mediated T cell B cell collaboration results in long-lasting germinal center formation and production of long-lived B cells that can later carry the virus to the mammary gland epithelium. Later in life it can induce transformation of mammary gland epithelium by integrating close to proto-oncogenes leading to their overexpression. Genes encoding proteins of the Wnt-pathway are preferential targets. This review will put these effects in the context of a normal immune response and summarize important facts on MMTV biology.

Streptococcal mitogenic exotoxin, SmeZ, is the most susceptible M1T1 streptococcal superantigen to degradation by the streptococcal cysteine protease, SpeB.

Superantigens (SAgs) play an important role in the pathogenesis of severe invasive infections caused by Group A Streptococcus (GAS). We had shown earlier that the expression of streptococcal cysteine protease SpeB results in partial loss of the immune-stimulating activity of the native secreted GAS SAgs, namely the streptococcal pyrogenic exotoxins produced by the globally disseminated M1T1 GAS strain, associated with invasive infections worldwide. In this study, we examined the susceptibility of each of the M1T1 recombinant SAgs to degradation by rSpeB. Whereas SmeZ was degraded completely within 30 min of incubation with rSpeB, SpeG, and SpeA were more resistant and SpeJ was completely unaffected by the proteolytic effects of this protease. Proteomic analyses demonstrated that the order of susceptibility of the M1T1 SAgs to SpeB proteolysis is unaltered when they are present in a mixture that reflects their native physiological status. As expected, the degradation of SmeZ abolished its immune stimulatory activity. In silico sequence disorder and structural analyses revealed that SmeZ, unlike the three other structurally related SAgs, possesses a putative SpeB cleavage site within an area of the protein likely to be exposed to the surface. The study provides evidence for the effect of subtle structural differences between highly similar SAgs on their biological activity.

Crystal structure of staphylococcal enterotoxin I (SEI) in complex with a human major histocompatibility complex class II molecule.

Superantigens are bacterial or viral proteins that elicit massive T cell activation through simultaneous binding to major histocompatibility complex (MHC) class II and T cell receptors. This activation results in uncontrolled release of inflammatory cytokines, causing toxic shock. A remarkable property of superantigens, which distinguishes them from T cell receptors, is their ability to interact with multiple MHC class II alleles independently of MHC-bound peptide. Previous crystallographic studies have shown that staphylococcal and streptococcal superantigens belonging to the zinc family bind to a high affinity site on the class II beta-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the beta-chain is polymorphic and the MHC-bound peptide forms part of the binding interface. To understand how zinc-dependent superantigens recognize MHC, we determined the crystal structure, at 2.0 A resolution, of staphylococcal enterotoxin I bound to the human class II molecule HLA-DR1 bearing a peptide from influenza hemagglutinin. Interactions between the superantigen and DR1 beta-chain are mediated by a zinc ion, and 22% of the buried surface of peptide.MHC is contributed by the peptide. Comparison of the staphylococcal enterotoxin I.peptide.DR1 structure with ones determined previously revealed that zinc-dependent superantigens achieve promiscuous binding to MHC by targeting conservatively substituted residues of the polymorphic beta-chain. Additionally, these superantigens circumvent peptide specificity by engaging MHC-bound peptides at their conformationally conserved N-terminal regions while minimizing sequence-specific interactions with peptide residues to enhance cross-reactivity.