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 β-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the β-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 2.0Å 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 β-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 β-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. 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 β-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the β-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 2.0Å 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 β-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 β-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. Superantigens (SAGs) 4The abbreviations used are: SAG, superantigen; SEI, staphylococcal enterotoxin I; TCR, T cell receptor; TSST-1, toxic shock syndrome toxin-1; SPE, streptococcal pyrogenic exotoxin; CDR, complementarity-determining region; HA, influenza virus hemagglutinin; MHC, major histocompatibility complex; MBP, myelin basic protein.4The abbreviations used are: SAG, superantigen; SEI, staphylococcal enterotoxin I; TCR, T cell receptor; TSST-1, toxic shock syndrome toxin-1; SPE, streptococcal pyrogenic exotoxin; CDR, complementarity-determining region; HA, influenza virus hemagglutinin; MHC, major histocompatibility complex; MBP, myelin basic protein. are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin with the ability to activate up to 20% of all T cells, as compared with on the order of only 0.001% of T cells for conventional peptide antigens (hence the term "superantigen") (1Scherer M.T. Ignatowicz L. Winslow G.M. Kappler J.W. Marrack P. Annu. Rev. Cell Biol. 1993; 9: 101-128Crossref PubMed Scopus (220) Google Scholar, 2Li H. Llera A. Malchiodi E.L. Mariuzza R.A. Annu. Rev. Immunol. 1999; 17: 435-466Crossref PubMed Scopus (256) Google Scholar). SAGs activate T cells by simultaneously binding T cell receptors (TCRs) and MHC class II molecules, resulting in the massive release of inflammatory cytokines, such as interleukin-1, interleukin-2, tumor necrosis factor-α, and tumor necrosis factor-β (1Scherer M.T. Ignatowicz L. Winslow G.M. Kappler J.W. Marrack P. Annu. Rev. Cell Biol. 1993; 9: 101-128Crossref PubMed Scopus (220) Google Scholar). These host cytokines are believed to be responsible for the most severe consequences of SAG intoxication, including capillary leak, renal failure, acute respiratory distress, and death (3McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). The best-characterized group of SAGs belongs to the pyrogenic toxin SAG family, which currently numbers 22 members produced by Staphylococcus aureus and Streptococcus pyogenes (3McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar, 4Sundberg E.J. Li Y. Mariuzza R.A. Curr. Opin. Immunol. 2002; 14: 36-44Crossref PubMed Scopus (60) Google Scholar). These toxins include staphylococcal enterotoxins A through M (SEA to SEM, except there is no F), staphylococcal toxic shock syndrome toxin-1 (TSST-1), streptococcal superantigen (SSA), streptococcal mitogenic exotoxin Z (SMEZ), and streptococcal pyrogenic exotoxins A (SPEA), SPEC, SPEG, SPEH, and SPEJ (5Bohach G.A. Fast D.J. Nelson R.D. Schlievert P.M. Crit. Rev. Microbiol. 1990; 17: 251-272Crossref PubMed Scopus (410) Google Scholar, 6Kotb M. Curr. Opin. Microbiol. 1998; 1: 56-65Crossref PubMed Scopus (40) Google Scholar, 7Dinges M.M. Orwin P.M. Schlievert P.M. Clin. Microbiol. Rev. 2000; 13: 16-34Crossref PubMed Scopus (1276) Google Scholar, 8McCormick J.K. Schlievert P.M. Fischetti V. Novick R. Ferretti J. Portnoy D. Rood J. Gram Positive Pathogens. American Society for Microbiology, Washington, D. C.2000: 43-52Google Scholar). These proteins are among the most potent pyrogens known and are capable of inducing toxic shock syndrome, an acute onset illness characterized by high fever and hypotension that can lead to multiple organ failure and lethal shock (3McCormick J.K. Yarwood J.M. Schlievert P.M. Annu. Rev. Microbiol. 2001; 55: 77-104Crossref PubMed Scopus (563) Google Scholar). Because of their extreme virulence and the ease with which they can be produced and disseminated, bacterial SAGs have been identified as category B agents of bioterrorism by the U. S. Centers for Disease Control and Prevention. 5Bioterrorism Agents/Diseases, U. S. Centers for Disease Control and Prevention, Atlanta, GA.5Bioterrorism Agents/Diseases, U. S. Centers for Disease Control and Prevention, Atlanta, GA. All known bacterial SAGs share a characteristic three-dimensional structure consisting of an N-terminalβ-barrel domain and a C-terminal β-grasp domain (2Li H. Llera A. Malchiodi E.L. Mariuzza R.A. Annu. Rev. Immunol. 1999; 17: 435-466Crossref PubMed Scopus (256) Google Scholar). Despite this common architecture, the complexes formed between SAGs and MHC molecules are structurally diverse, considerably more so than SAG·TCR complexes (4Sundberg E.J. Li Y. Mariuzza R.A. Curr. Opin. Immunol. 2002; 14: 36-44Crossref PubMed Scopus (60) Google Scholar) Thus, x-ray crystallographic and binding studies have shown that MHC class II molecules possess two independent binding sites for bacterial SAGs: 1) a low affinity site (KD ∼ 10–5 m) on the conserved α-chain; and 2) a zinc-dependent, high affinity site (KD ∼ 10–7 m) on the polymorphic β-chain. Based on sequence analysis, SAGs may be divided into three groups according to how they bind MHC: 1) those that bind only the MHC α-chain through the low affinity site (SEB, SEC1–3, SEG, TSST-1, SPEA, SSA); 2) those that bind only the MHC β-chain through the zinc-dependent, high affinity site (SEH, SEI, SEJ, SEK, SEL, SEM, SPEC, SPEG, SPEH, SPEJ, SMEZ); and 3) those that cross-link two MHC molecules by simultaneously binding the α-chain of one MHC and the β-chain of another MHC through the low and high affinity sites, respectively (SEA, SED, SEE). A unique feature of bacterial SAGs, which is a major contributor to their toxicity, is the ability of individual SAGs to bind different MHC class II molecules, largely irrespective of the sequence of the MHC-bound peptide. In this way, SAGs maximize TCR-MHC interactions and the resulting T cell activation. For SAGs such as SEB and SEC3, which bind to the low affinity site on the class II α-chain, recognition of multiple MHC alleles by a single toxin is readily explained by crystal structures of these SAGs bound to HLA-DR1, a human class II molecule (10Jardetzky T.S. Brown J.H. Gorga J.C. Stern L.J. Urban R.G. Chi Y.I. Stauffacher C. Strominger J.L. Wiley D.C. Nature. 1994; 368: 711-718Crossref PubMed Scopus (498) Google Scholar, 11Sundberg E.J. Andersen P.S. Schlievert P.M. Karjalainen K. Mariuzza R.A. Structure (Camb.). 2003; 11: 1151-1161Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In these complexes, the N-terminal domain of SEB or SEC3 contacts the α1 domain of DR1, away from the peptide-binding groove. Because the DR α-chain is nonpolymorphic and because the SAG does not contact the MHC-bound peptide, the result is promiscuous MHC recognition. By contrast, the structural basis for cross-recognition of MHC by zinc-dependent SAGs is much less apparent, despite the available structures of two such SAGs, SEH and SPEC, bound to HLA-DR1 and HLA-DR2a, respectively (12Petersson K. Hakansson M. Nilsson H. Forsberg G. Svensson L. Lijas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (84) Google Scholar, 13Li Y. Li H. Dimassi N. McCormick J.K. Martin R. Schuck P. Schlievert P.M. Mariuzza R.A. Immunity. 2001; 14: 93-104Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In these complexes, the C-terminal domain of the SAG contacts the β1 domain of HLA-DR, which is polymorphic, as well as the MHC-bound peptide. Indeed, the peptide accounts for ∼25% of the surface area of the MHC molecule buried by SEH or SPEC, similar to TCR·peptide·MHC complexes, in which the TCR exhibits exquisite specificity for both peptide and MHC (14Rudolph M.G. Stanfield R.L. Wilson I.A. Annu. Rev. Immunol. 2006; 24: 419-466Crossref PubMed Scopus (892) Google Scholar). To better understand MHC recognition by zinc-dependent SAGs, we determined the crystal structure of staphylococcal enterotoxin I (SEI) bound to HLA-DR1 bearing a peptide from influenza virus hemagglutinin (HA-(306–318)). SEI, which is encoded in the egc operon of S. aureus (15Jarraud S. Peyrat M.A. Lim A. Tristan A. Bes M. Mougel C. Etienne J. Vandenesch F. Bonneville M. Lina G. J. Immunol. 2001; 166: 669-677Crossref PubMed Scopus (422) Google Scholar), is associated with both menstrual and nonmenstrual toxic shock syndrome, in addition to food poisoning (16Banks M.C. Kamel N.S. Zabriskie J.B. Larone D.H. Ursea D. Posnett D.N. J. Infect. Dis. 2003; 187: 77-86Crossref PubMed Scopus (32) Google Scholar) and various veterinary diseases (17Omoe K. Ishikawa M. Shimoda Y. Hu D.L. Ueda S. Shinagawa K. J. Clin. Microbiol. 2002; 40: 857-862Crossref PubMed Scopus (307) Google Scholar). Comparison of the SEI·HA·HLA-DR1 structure with those of SEH and SPEC bound to MHC class II (12Petersson K. Hakansson M. Nilsson H. Forsberg G. Svensson L. Lijas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (84) Google Scholar, 13Li Y. Li H. Dimassi N. McCormick J.K. Martin R. Schuck P. Schlievert P.M. Mariuzza R.A. Immunity. 2001; 14: 93-104Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) reveals how zinc-dependent SAGs circumvent peptide specificity and how they target key residues of the polymorphic class II β-chain to achieve promiscuous binding to peptide·MHC. Expression and Purification of SEI—The sei gene was cloned and expressed in Escherichia coli as described previously (18Fernández M.M. De Marzi M.C. Berguer P. Burzyn D. Langley R.J. Piazzon I. Mariuzza R.A. Malchiodi E.L. Mol. Immunol. 2006; 43: 927-938Crossref PubMed Scopus (26) Google Scholar). Briefly, sei from clinically isolated S. aureus strain Fc30 (AAX84810) was cloned into the BamHI/EcoRI cloning site of the bacterial expression vector pET32a. 3C, which encodes thioredoxin and a 3C protease recognition sequence (19Walker P.A. Leong L.E. Ng P.W. Tan S.H. Waller S. Murphy D. Porter A.G. Bio/Technology. 1994; 12: 601-605Crossref PubMed Scopus (134) Google Scholar) directly upstream of the BamHI site. Six histidine-encoding triplets were grafted onto the 3′ terminus of the sei gene to facilitate purification of the thioredoxin-SEI fusion protein. For expression, transformed Origami (DE3) cells (Novagen) were grown at 30 °C in LB medium containing 100 μg/ml ampicillin and 30 μg/ml chloramphenicol. Bacteria were induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside at an absorbance of 1.0 at 600 nm. After induction, the cultures were incubated for 4 h with constant shaking. SEI was expressed as a soluble cytoplasmic fusion protein (20Langley R. Wines B. Willoughby N. Basu I. Proft T. Fraser J. J. Immunol. 2005; 174: 2926-2933Crossref PubMed Scopus (179) Google Scholar). The protein was purified using a Ni2+ chelate adsorbent (Qiagen), cleaved with 3C protease (kindly provided by Dr. R. Langley) and resubjected to Ni2+ affinity chromatography to separate SEI from thioredoxin. SEI was further purified using a Mono S cation exchange column (Amersham Biosciences). Expression and Purification of HLA-DR1—HLA-DR1 was produced by in vitro folding from bacterial inclusion bodies as described (21Frayser M. Sato A.K. Xu L. Stern L.J. Protein Expr. Purif. 1999; 15: 105-114Crossref PubMed Scopus (87) Google Scholar). Briefly, plasmids encoding the HLA-DR1 α-chain (DRA*0101) and β (DRB1*0101) chain were transformed separately into E. coli BL21(DE3) cells (Stratagene). Bacteria were grown at 37 °C to an absorbance of 0.6–0.7 at 600 nm, and 1 mm isopropyl-β-d-thiogalactopyranoside was added. Inclusion bodies were washed extensively and the subunits purified under denaturing and reducing conditions using an HQ50 anion exchange column (PerSeptive Biosystems). Yields of DR1 α- and β-subunits were 16 and 20 mg/liter of culture medium, respectively. Purified subunits were diluted dropwise with constant stirring to a final concentration of 50 μg/ml into a folding solution of 20 mm Tris-HCl (pH 8.5), 25% (w/v) glycerol, 0.5 mm EDTA, 3 mm reduced glutathione, and 0.3 mm oxidized glutathione and kept at 4 °C for 2 days in the presence of 1 μm HA-(306–318) peptide (PKYVKQNTLKLAT). Recombinant HA·HLA-DR1 was purified from the folding mixture using a Mono Q anion exchange column (Amersham Biosciences) equilibrated with 20 mm Tris-HCl (pH 8.0) and developed with a linear NaCl gradient. The protein eluted as a single peak at 0.15 m NaCl. Crystallization and Data Collection—HLA-DR1·HA and SEI were premixed in a 1:1 molar ratio, and the complex was purified with a Superdex S-200 column (Amersham Biosciences) prior to crystallization. Crystals were grown at room temperature in hanging drops by mixing 1 μl of protein solution at 10 mg/ml in 10 mm Hepes and 15 mm NaCl (pH 7.5) with 1 μlof reservoir solution containing 10% (v/v) dioxane, 1.2 mm (NH4)2SO4, and 100 mm Hepes (pH 7.5). Large, but twinned, pyramidal crystals grew over a period of 2 weeks. Single crystals were obtained by microseeding at a protein concentration of 7 mg/ml. For data collection, crystals were cryoprotected by brief soaking in mother liquor containing 15% (v/v) glycerol and flash-cooled in a nitrogen stream. X-ray diffraction data to 2.0 Å resolution were recorded in-house at 110 K using an R-axis IV2+ image plate detector equipped with Osmic mirrors and mounted on a Rigaku rotating anode Cu-Kα x-ray generator. The data were processed and scaled with CrystalClear (22Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1410) Google Scholar). Data collection statistics are summarized in Table 1.TABLE 1Data collection and structure refinement statisticsData collectionResolution range (Å)40.0-2.0Space groupC2Cell parameters (Å, °)a = 150.40, b = 99.93, c = 72.92 β = 92.0Unique reflectionsaValues in parentheses are statistics of the highest resolution shell (2.07-2.00 Å).72,148 (7089)Completeness (%)aValues in parentheses are statistics of the highest resolution shell (2.07-2.00 Å).99.2 (98.0)Rmerge (%)aValues in parentheses are statistics of the highest resolution shell (2.07-2.00 Å).,bRmerge = Σ|Ij — 〈I 〉|ΣIj, where Ij is the intensity of an individual reflection and 〈I 〉 is the average intensity of that reflection.7.5 (37.8)I/σ(I)9.7 (3.3)Redundancy4.05 (3.93)RefinementResolution range (Å)40.0-2.0Rcryst (%)cRcryst = Σ∥Fo| — |Fc∥|Σ|Fo|, where Fc is the calculated structure factor. Rfree is same as for Rcryst but is calculated for a randomly selected 4.0% of reflections not included in the refinement.21.3Rfree (%)cRcryst = Σ∥Fo| — |Fc∥|Σ|Fo|, where Fc is the calculated structure factor. Rfree is same as for Rcryst but is calculated for a randomly selected 4.0% of reflections not included in the refinement.25.2No. of reflections used69,190No. of reflections in Rfree set2,949No. of non-hydrogen protein atoms4,859No. of SO42- ions4No. of Zn2+ ions1No, of Hepes molecules1No. of dioxane molecules2No. of water molecules460r.m.s. deviation bond lengths (Å)dr.m.s., root mean square.0.012r.m.s. deviation bond angles (°)dr.m.s., root mean square.1.45Average temperature factors (Å2)Protein main chain atoms33.6Protein side chain atoms34.2Waters35.6Temperature factor from Wilson plot (Å2)29.4Ramachandran plot statisticsMost favored (%)90.8Additional allowed (%)8.8Generously allowed (%)0.4a Values in parentheses are statistics of the highest resolution shell (2.07-2.00 Å).b Rmerge = Σ|Ij — 〈I 〉|ΣIj, where Ij is the intensity of an individual reflection and 〈I 〉 is the average intensity of that reflection.c Rcryst = Σ∥Fo| — |Fc∥|Σ|Fo|, where Fc is the calculated structure factor. Rfree is same as for Rcryst but is calculated for a randomly selected 4.0% of reflections not included in the refinement.d r.m.s., root mean square. Open table in a new tab Structure Determination and Refinement—The structure of the SEI·HA·HLA-DR1 complex was solved by molecular replacement using Phaser (23McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1590) Google Scholar). The search models consisted of SEA (24Sundstrom M. Hallen D. Svensson A. Schad E. Dohlsten M. Abrahmsen L. J. Biol. Chem. 1996; 271: 2212-2216Google Scholar) (Protein Data Bank accession code 1SXT) and HA-(306–318)·HLA-DR1 (25Stern L. Brown J. Jardetzky T. Gorga J. Urban R. Strominger J. Wiley D. Nature. 1994; 368: 215-221Crossref PubMed Scopus (1431) Google Scholar) (1DLH). Only one SEI·HA·HLA-DR1 complex molecule was found in the asymmetric unit, corresponding to a Matthews coefficient of 3.72 (solvent content ∼67%). The molecular replacement solution was refined with Refmac 5.2 (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 53: 240-255Crossref Scopus (13708) Google Scholar), and the model was adjusted manually with XtalView (27McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2016) Google Scholar) based on σA-weighted Fo – Fc and 2Fo – Fc electron density maps. TLS parameters were refined as well as temperature (B) factors. The final model comprises residues 4–216 of SEI, 4–181 of DR1-α, 1–190 of DR1-β, and 306–318 of HA and 460 water molecules. The model also contains one zinc ion, four sulfate ions, two dioxane molecules, and one Hepes molecule, resulting in a final Rcryst of 21.3% and Rfree of 25.2% at 2.0 Å resolution. The quality of the model was examined with PROCHECK (28Laskowski R. Moss D. Thornton J. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1071) Google Scholar) and Molprobity (29Davis I.W. Murray L.W. Richardson J.S. Richardson D.C. Nucleic Acids Res. 2004; 32: W615-W619Crossref PubMed Scopus (791) Google Scholar). Refinement statistics are summarized in Table 1. Atomic coordinates and structure factors for the SEI·HA·HLA-DR1 complex have been deposited in the Protein Data Bank under accession code 2G9H. Overview of the Complex—The structure of SEI bound to HA-(306–318)·HLA-DR1 was determined by molecular replacement to 2.0 Å resolution (Table 1). The overall structure of the complex is shown in Fig. 1A. All residues in the interface between SAG and MHC molecules show unambiguous electron density. In addition, all SEI residues are well defined, including those forming the β4-β5 loop (see below). In the complex, all contacts made by SEI are to the DR1 β-chain and HA peptide; there are no interactions between the DR1 α-chain and the SAG. The β-grasp motif of the C-terminal portion of SEI contacts the α-helix of the β1 domain of HLA-DR1, as well as the N-terminal portion of HA-(306–318) (Fig. 1B). The interaction between SEI and the DR1 β-chain is mediated in part by a zinc ion, for which identity was confirmed by atomic absorption spectroscopy (data not shown). The importance of Zn2+ to complex stabilization is supported by the finding that the addition of EDTA abolishes binding of SEI to HLA-DR1 in solution (18Fernández M.M. De Marzi M.C. Berguer P. Burzyn D. Langley R.J. Piazzon I. Mariuzza R.A. Malchiodi E.L. Mol. Immunol. 2006; 43: 927-938Crossref PubMed Scopus (26) Google Scholar). However, the occupancy of Zn2+ in the crystal is surprisingly low. In the final refined model, its occupancy was set to 0.3, which gave a temperature factor of 30.7 Å2, very close to the average main chain atom B-value of 33.6 Å2 for the protein molecules (Table 1). The low zinc occupancy could be explained by traces of EDTA in the protein solution used for crystallization, because EDTA was present throughout the preparation of HLA-DR1 except at the final dialysis stage. Residual EDTA could have stripped Zn2+ from SEI, either before or after crystal formation. Although EDTA eliminates detectable binding of SEI to HLA-DR1 during gel filtration (18Fernández M.M. De Marzi M.C. Berguer P. Burzyn D. Langley R.J. Piazzon I. Mariuzza R.A. Malchiodi E.L. Mol. Immunol. 2006; 43: 927-938Crossref PubMed Scopus (26) Google Scholar), it is likely that the SAG retains some affinity for MHC, even in the absence of Zn2+. Under the high protein concentrations in crystallization drops, low affinities often suffice for co-crystallization. Alternatively, even if each complex molecule in the crystal initially contained Zn2+, its partial removal by EDTA would not necessarily disrupt the SEI·HA·DR1 complex, because lattice contacts could compensate for reductions in affinity. Conformation of the β4-β5 Loop and Implications for TCR Binding—In nearly all previously reported SAG structures, both free and bound to MHC or TCR ligands, little or no electron density could be observed for residues connecting β-strands 4 and 5 of the N-terminal domain (10Jardetzky T.S. Brown J.H. Gorga J.C. Stern L.J. Urban R.G. Chi Y.I. Stauffacher C. Strominger J.L. Wiley D.C. Nature. 1994; 368: 711-718Crossref PubMed Scopus (498) Google Scholar, 24Sundstrom M. Hallen D. Svensson A. Schad E. Dohlsten M. Abrahmsen L. J. Biol. Chem. 1996; 271: 2212-2216Google Scholar, 30Schad E.M. Zaitseva I. Zaitsev V.N. Dohlsten M. Kalland T. Schlievert P.M. Ohlendorf D.H. Svensson L.A. 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Yang J. Kerzic M.C. Guan R. Kieke M.C. Kranz D.M. Mariuzza R.A. Sundberg E.J. Structure (Camb.). 2005; 13: 1775-1787Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), implying flexibility. In the SEI·HA·DR1 complex, by contrast, the β4-β5 loop of SEI (residues 67–75) is well ordered (Fig. 2A), even though it lacks the disulfide bond typical of other SAGs. Sequence alignments show that this loop is 1–6 residues shorter in SEI than in most SAGs (Fig. 3), which could explain why it adopts a defined conformation in the SEI·HA·DR1 complex (the structure of unbound SEI is unknown). Alternatively, lattice contacts with a neighboring complex molecule in the crystal (which include several hydrogen bonds between loop residues Cys73 and Ser75 and a symmetry-related DR1 molecule) may account for the ordering of the β4-β5 loop in the SEI·HA·DR1 structure.FIGURE 3Structure-based sequence alignment of the zinc family of superantigens. Secondary structure elements for SEI (top) are denoted by squiggles (α-helices and 310 helices (η)) and arrows (β-strands); these are numbered according to their order of appearance in the sequences. Numbers at the top refer to SEI residues. White characters on a red background show strictly conserved residues. Residues that are well conserved are drawn in red and framed in blue. The remaining residues are black. Triangles above the SEI sequence mark residues involved in interactions with HA·HLA-DR1 in the crystal structure. The three residues involved in Zn2+ coordination in SEI are marked with green triangles; other interacting residues are marked with blue triangles. Sequence alignments were performed with ClustalW, and the figure was generated using ESPript.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The conformation and amino acid sequence of the β4-β5 loop may influence the Vβ binding specificity of SEI and other SAGs. Thus, the structure of SPEC in complex with human Vβ2.1 showed that the β4-β5 loop makes a substantial contribution to the binding interface (38Sundberg E.J. Li H. Llera A.S. McCormick J.K. Tormo J. Schlievert P.M. Karjalainen K. Mariuzza R.A. Structure (Lond.). 2002; 10: 687-699Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Like SPEC, the five other known zinc-dependent SAGs from Streptococcus (SPEG, SPEH, SPEJ, SMEZ1, and SMEZ2) also stimulate T cells expressing Vβ2.1 (39Proft T. Fraser J.D. Clin. Exp. Immunol. 2003; 133: 229-306Crossref Scopus (343) Google Scholar). By contrast, no zinc-dependent SAG from Staphylococcus, including SEI, recognizes Vβ2.1. To help explain the lack of interaction between SEI and Vβ2.1, SEI was superposed onto SPEC in the SPEC·Vβ2.1 structure (38Sundberg E.J. Li H. Llera A.S. McCormick J.K. Tormo J. Schlievert P.M. Karjalainen K. Mariuzza R.A. Structure (Lond.). 2002; 10: 687-699Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In the docked complex (Fig. 2B), major steric clashes are evident between the β4-β5 loop of SEI and the first and second complementarity-determining regions (CDR1 and CDR2) of Vβ2.1. These clashes arise from the very different orientations of the β4-β5 loop in SEI and SPEC, underscoring the importance of this loop in determining Vβ-binding specificity. Structure of the Interface and Interactions with Zinc and Peptide·MHC—The complex of SEI with HA·DR1 buries 1163 Å2 of surface area as calculated with Areaimol in the CCP4 program suite (40Collaborative Computational Project, Number 4 (CCP4) Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19664) Google Scholar), of which 596 Å2 is contributed by peptide·MHC and 567 Å2 by the SAG. By comparison, the total buried surfaces in the SEH·HA·DR1 and SPEC·MBP·DR2a complexes are considerably greater: 1465 and 1628 Å2, respectively (12Petersson K. Hakansson M. Nilsson H. Forsberg G. Svensson L. Lijas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (84) Google Scholar, 13Li Y. Li H. Dimassi N. McCormick J.K. Martin R. Schuck P. Schlievert P.M. Mariuzza R.A. Immunity. 2001; 14: 93-104Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). For the SEI·HA·DR1 complex, 22% (132 Å2) of the buried surface of peptide·MHC is contributed by the MHC-bound peptide, compared with 28% (208 Å2) for the SEH·HA·DR1 complex and 34% (265 Å2) for the SPEC·MBP·DR2a complex. No major rearrangeme