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• W2003262553 abstract "The previously determined crystal structure of the superantigen staphylococcal enterotoxin C2 (SEC2) showed binding of a single zinc ion located between the N- and C-terminal domains (Papageorgiou, A. C., Acharya, K. R., Shapiro, R., Passalacqua, E. F., Brehm, R. D., and Tranter, H. S. (1995) Structure 3, 769-779). Here we present the crystal structure of SEC2 determined to 2.0 Å resolution in the presence of additional zinc. The structure revealed the presence of a secondary zinc-binding site close to the major histocompatibility complex (MHC)-binding site of the toxin and some 28 Å away from the primary zinc-binding site of the toxin found in previous studies. T cell stimulation assays showed that varying the concentration of zinc ions present affected the activity of the toxin and we observed that high zinc concentrations considerably inhibited T cell responses. This indicates that SEC2 may have multiple modes of interaction with the immune system that are dependent on serum zinc levels. The potential role of the secondary zinc-binding site and that of the primary one in the formation of the TCR·SEC2·MHC complex are considered, and the possibility that zinc may regulate the activity of SEC2 as a toxin facilitating different T cell responses is discussed. The previously determined crystal structure of the superantigen staphylococcal enterotoxin C2 (SEC2) showed binding of a single zinc ion located between the N- and C-terminal domains (Papageorgiou, A. C., Acharya, K. R., Shapiro, R., Passalacqua, E. F., Brehm, R. D., and Tranter, H. S. (1995) Structure 3, 769-779). Here we present the crystal structure of SEC2 determined to 2.0 Å resolution in the presence of additional zinc. The structure revealed the presence of a secondary zinc-binding site close to the major histocompatibility complex (MHC)-binding site of the toxin and some 28 Å away from the primary zinc-binding site of the toxin found in previous studies. T cell stimulation assays showed that varying the concentration of zinc ions present affected the activity of the toxin and we observed that high zinc concentrations considerably inhibited T cell responses. This indicates that SEC2 may have multiple modes of interaction with the immune system that are dependent on serum zinc levels. The potential role of the secondary zinc-binding site and that of the primary one in the formation of the TCR·SEC2·MHC complex are considered, and the possibility that zinc may regulate the activity of SEC2 as a toxin facilitating different T cell responses is discussed. A number of toxins produced by Staphylococcus aureus and Streptococcus pyogenes, known as “superantigens,” are potent inducers of T cell activity. In contrast to conventional antigens, superantigens bind to MHC 1The abbreviations used are: MHC, major histocompatibility complex; CHO, Chinese hamster ovary; r.m.s., root mean square; SAg, superantigen; SE, staphylococcal enterotoxins; Spe, streptococcal pyrogenic exotoxin; TCR, T cell receptor; TSST-1, toxic shock syndrome toxin-1. class II molecules on antigen-presenting cells outside the antigen binding cleft and are presented as unprocessed proteins to T cell receptors carrying particular Vβ chains. Very low concentrations of superantigens are able to activate a large amount of resting T cells, thereby inducing massive cytokine release (1.Marrack P. Kappler J. Science. 1990; 248: 1335-1341Crossref PubMed Scopus (526) Google Scholar, 2.Herman A. Kappler J.W. Marrack P. Pullen A.M. Annu Rev. Immunol. 1991; 9: 745-772Crossref PubMed Scopus (794) Google Scholar), which may result in acute systemic illness and clinical shock (3.Todd J. Fishaut M. Kapral F. Welch T. Lancet. 1978; 2: 1116-1118Abstract PubMed Scopus (811) Google Scholar). Members of the superantigen family include the staphylococcal enterotoxins (SEs) A, B, C, D, E, and H, toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxins (Spes) A, B, and C. Amino acid sequence comparison shows that the staphylococcal enterotoxins fall into two main groups with SEA, -D, -E, and -H comprising one group and SEB and the SECs, the second group. The SECs can be classified further into at least three serotypes (C1 to C3) depending on minor epitope differences. TSST-1 shows little sequence homology (less than 28%) with the SEs (4.Blomster-Hautamaa D.A. Schlievert P.M. Methods Enzymol. 1988; 165: 37-43Crossref PubMed Scopus (77) Google Scholar), but it is more similar to SpeC. Crystallographic studies have led to the elucidation of the crystal structures of SEA (5.Schad E.M. Zaitseva I. Zaitsev V.N. Dohlsten M. Kalland T. Schlievert P.M. Ohlendorf D.H. Svensson L.A. EMBO J. 1995; 14: 3292-3301Crossref PubMed Scopus (180) Google Scholar), SEC2 (6.Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure. 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), SEB (7.Papageorgiou A.C. Tranter H.S. Acharya K.R. J. Mol. Biol. 1998; 277: 61-79Crossref PubMed Scopus (103) Google Scholar), TSST-1 (8.Acharya K.R. Passalacqua E.F. Jones E.Y. Harlos K. Stuart D.I. Brehm R.D. Tranter H.S. Nature. 1994; 367: 94-97Crossref PubMed Scopus (144) Google Scholar), SED (9.Sundstrom M. Abrahmsen L. Antonsson P. Mehindate K. Mourad W. Dohlsten M. EMBO J. 1996; 15: 6832-6840Crossref PubMed Scopus (94) Google Scholar), and SpeA1 (10.Papageorgiou A.C. Collins C.M. Gutman D.M. Kline J.B. O'Brien S.M. Tranter H.S. Acharya K.R. EMBO J. 1999; 18: 9-21Crossref PubMed Scopus (76) Google Scholar) and have revealed a common fold shared among the superantigens. Moreover, the crystal structures of TSST-1 and SEB in the presence of a MHC class II molecule and SEC2/SEC3 in complex with a TCR Vβ chain (11.Fields B.A. Malchiodi E.L. Li H. Ysern X. Stauffacher C.V. Schlievert P.M. Karjalainen K. Mariuzza R.A. Nature. 1996; 384: 188-192Crossref PubMed Scopus (259) Google Scholar) have been reported. From these studies, it is now well established that despite sequence and structural similarity, each superantigen appears to have adopted a different mechanism in forming the MHC·superantigen·TCR tri-molecular complex. The presence of one (SEA, SEC2, SpeA1) or two (SED, SpeC) zinc-binding sites plays a central role in this diversity. The crystal structure of SEC2 has been determined previously in two different space groups: tetragonal P43212 (crystal form I (6.Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure. 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar)) and monoclinic P21 (crystal form II (12.Swaminathan S. Furey W. Pletcher J. Sax M. Nat. Struct. Biol. 1995; 2: 680-686Crossref PubMed Scopus (56) Google Scholar)) at 2.0 and 2.7 Å, respectively. A striking difference between these two SEC2 structures is the presence of a zinc-binding site found in crystal form I but not in crystal form II (we refer to this primary site as Zn I). Here we report a third crystal form (form III) of this toxin grown in the presence of 2.5 mm zinc acetate. The structure revealed the binding of a second zinc atom some 28 Å away from the primary site (we refer to this secondary site as II). It is shown that the presence of zinc ions in the crystallization medium results in conformational changes and the formation of a secondary zinc-binding site close to the normal MHC-binding site of the toxin. In addition, the recent crystal structures of SpeC and SEH in complex MHC class II molecules via their zinc-binding sites have demonstrated that a single zinc ion can act as a high affinity binding site for MHC class II molecules (13.Li Y. Li H. Dimasi 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 (110) Google Scholar, 14.Petersson K. Hakansson M. Nilsson H. Forsberg G. Svensson L.A. Liljas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (86) Google Scholar). Thus, the presence of a secondary zinc site may have implications for the binding of the MHC class II molecules, suggesting different mechanisms of T cell activation under variable zinc concentrations, which might be linked to the function of SEC2. Moreover, it could guide the efforts to control the effects of enterotoxins on the immune system by controlling the zinc concentrations. T cell Stimulation—Peripheral blood mononuclear cells were obtained from healthy donors and isolated by Ficoll density gradient centrifugation. The buoyant layer was removed, and following several washes in phosphate-buffered saline to remove platelets, purified resting T cells were isolated as described previously (15.Sansom D.M. Wilson A. Boshell M. Lewis J. Hall N.D. Immunology. 1993; 80: 242-247PubMed Google Scholar). T cells were stimulated by the addition of HLA·DR4·CD80 transfectants the presence or absence of 100 ng/ml SEC. Assays were carried out at a range of concentrations of ZnCl2 or MgCl2 as indicated. 1 × 105 T cells were incubated at 37° C with 2.5 × 104 CHO transfectants in complete medium (RPMI 1640 plus 10% fetal calf serum and penicillin/streptomycin) in 200-μl final volumes in 96-well plates. After 72 h, 1 μCi of [3H]thymidine was added overnight, and proliferation was assessed by liquid scintillation counting (Packard Top-count). All stimulations were performed in triplicate. The data presented are the mean values from a representative experiment. Standard errors of triplicate samples are shown. Statistical comparisons were made using Student's t test. The experiment shown is representative of four others performed. Transfectants—CHO cells were transfected with cDNAs encoding human HLADR4 *0401β and HLADRα chains under the control of a cytomegalovirus promoter. These transfectants also expressed the human CD80 molecule to provide co-stimulation for T cell proliferation. Cells were grown and selected as described previously (15.Sansom D.M. Wilson A. Boshell M. Lewis J. Hall N.D. Immunology. 1993; 80: 242-247PubMed Google Scholar). Transfectants (1 × 106 cells/ml) were fixed prior to use by treating with 0.025% glutaraldehyde for 2 min and then washed three times in complete medium. Parental CHO cells not expressing HLA-DR molecules were used as a controls and did not stimulate proliferative responses. Crystallization and X-ray Data Collection—SEC2 was purified by the method of dye affinity chromatography as described previously by Passalacqua et al. (16.Passalacqua E.F. Brehm R.D. Acharya K.R. Tranter H.S. J. Mol. Biol. 1993; 233: 170-172Crossref PubMed Scopus (8) Google Scholar) and lyophilized. Crystals were grown using the hanging drop vapor diffusion method at 16 °C. The lyophilized protein was dissolved in MilliQ water to a concentration of ≈10 mg/ml. Four microliters of a reservoir solution containing 17-20% polyethylene glycol 6000, 0.02% sodium azide, 0.1-10 mm zinc acetate, and 0.1 m cacodylate buffer (pH 6.5) were mixed with an equal volume of the protein stock solution on siliconized coverslips. The best crystals, grown from 18% polyethylene glycol 6000 and 2.5 mm zinc acetate, were used for data collection. Although lower concentrations of zinc (as low as 0.1 mm) can also produce crystals, these crystals were very small and thin and were not suitable for x-ray analysis. The crystals belong to the orthorhombic space group P212121, with unit cell dimensions a = 42.54 Å, b = 44.69 Å, c = 131.42 Å and one SEC2 molecule/crystallographic asymmetric unit. Approximately 40% of the crystal volume is occupied by solvent (Matthews coefficient, Vm = 2.1 Å3/D). X-ray diffraction data were collected on station PX 9.5 at the Synchrotron Radiation Source, Daresbury, UK, and BW7B in EMBL (c/o DESY, Hamburg). Three data sets were collected at different times. The oscillation range was 1.5° and the exposure times were 20 s (Hamburg) and 60 s (Daresbury). The data were processed, scaled, and merged using the HKL package (17.Otwinowski Z. DENZO: An Oscillation Data Processing Programme for Macromolecular Crystallography. Yale University, New Haven, CT1993Google Scholar, 18.Otwinowski Z. SCALEPACK: Software for the Scaling Together of Integrated Intensities Measured on a Number of Separate Diffraction Images. Yale University, New Haven, CT1993Google Scholar) to give a master data set to 2.0 Å with an Rmerge of 7.8% and an overall completeness of 91.4%. Details of the data processing and refinement statistics are given in Table I.Table IData processing and refinement statisticsData set IData set IIUnit cell a = 42.50, b = 44.54, c = 131.65 (Å)Space group P212121Resolution range (Å)20.0—2.440.0—2.0Wavelength (Å)0.9199, 1.41480.8838No. of measurements91,59880,816No. of unique reflections10,05416,364Overall completeness (%)95.8 (91.7)aOutermost shell, 2.49—2.4 Å91.4(72.1)bOutermost shell, 2.06—2.0 ÅRmerge (%)cRmerge = Σ( | Ij — 〈I〉 | )/Σ〈I〉, where Ij is the observed intensity of reflection j and 〈I〉 is the average intensity of multiple observations7.1 (19.0)aOutermost shell, 2.49—2.4 Å7.8 (29.7)bOutermost shell, 2.06—2.0 ÅResolution range used in refinement40.0—2.0No. of reflections used in refinement16,330Rcryst (%)dRcryst = Σ |Fo| — |Fc|/Σ|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively19.82Rfree (%)e5% of the data used for the calculation of Rfree were randomly excluded from the refinement22.54r.m.s. deviationBond lengths (Å)0.006Angles (°)1.18B-factors (Å2)Wilson20.7Main chain26.04Side chain26.78Zn I13.90Zn II20.68Water30.9a Outermost shell, 2.49—2.4 Åb Outermost shell, 2.06—2.0 Åc Rmerge = Σ( | Ij — 〈I〉 | )/Σ〈I〉, where Ij is the observed intensity of reflection j and 〈I〉 is the average intensity of multiple observationsd Rcryst = Σ |Fo| — |Fc|/Σ|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectivelye 5% of the data used for the calculation of Rfree were randomly excluded from the refinement Open table in a new tab Structure Determination and Refinement—The SEC2 structure of the new orthorhombic crystal form (form III) was determined by molecular replacement using the program AMoRe (19.Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar). The previously determined SEC2 structure (space group P43212; Papageorgiou et al. (6.Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure. 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar)) at 2.0 Å resolution was used as a search model. The cross-rotation search gave an unambiguous solution (correlation coefficient 29.5, 14σ above the mean), which still remained at the top of the solution list after the translation search. Examination of the solution after rigid body refinement in AMoRe showed no crystal-packing clashes. Initially refinement was carried out with the 2.4 Å data set with an initial Rcryst and Rfree (5% of the reflections set aside (20.Brünger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3872) Google Scholar)) of 32.8 and 33.9%, respectively. When the Rcryst and Rfree dropped to 22.1 and 28.5%, respectively, a new data set to 2.0 Å resolution was collected. Extension to 2.0 Å was accomplished in 0.1 Å steps using the simulated annealing protocol in X-PLOR (version 3.851 (21.Brünger A.T. X-PLOR: Version 3.1. A System for Crystallography and NMR. Yale University, New Haven, CT1992Google Scholar)). Reflections flagged for Rfree calculations in the 2.4 Å data set were carried over to the new data set. SigmaA-weighted 2 Fo - Fc and Fo - Fc maps were generated with the CCP4 suite of programs and examined manually using the program O (23.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 109-110Google Scholar). Water molecules within hydrogen bonding distances from suitable protein atoms were identified manually, and their positions were checked with the aid of 2 Fo - Fc maps (at 1σ) and Fo - Fc maps (at 3σ). Water molecules with temperature factors higher than 50 Å2 after one round of refinement were excluded from subsequent refinement. Refinement was concluded using the program CNS (crystallography NMR software), version 1.1 (22.Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) with iterative cycles of minimization, individual B-factor refinement, and simulated annealing followed by calculation of 2 Fo - Fc and Fo - Fc maps. Quality of the Structure—The final model, comprising 1851 protein atoms, 77 water molecules, and two zinc ions, has a crystallographic R-factor (Rcryst) of 19.82% and an Rfree of 22.54%. The r.m.s. deviation in bond lengths and bond angles is 0.006 Å and 1.18°, respectively. Examination of the Ramachandran plot with PROCHECK (24.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) showed 90.6% of non-glycine and non-proline residues to be in the most favored regions and none in the disallowed regions. Details of data processing and refinement statistics are shown in Table I. The mean coordinate errors derived from a plot of sigmaA versus (sinθ/λ)2 is 0.22 Å. Residues 98-106 from the disulfide loop have been assigned an occupancy of 0 due to high mobility. The unique feature of the SEC2 structure presented here is the presence of two zinc-binding sites. The primary zinc-binding site was reported previously as a strong feature of the difference electron map at 9σ level. The secondary zinc-binding site (reported here) is some 28 Å away from the primary site. Both zinc ions are characterized by low temperature factors (13.9 and 20.68 Å2) with the former one having similar temperature factor as that reported in crystal form I (6.Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure. 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Description of the Structure—The structure of SEC2 is similar to that of other superantigens of staphylococcal or streptococcal origin (25.Papageorgiou A.C. Acharya K.R. Structure. 1997; 5: 991-996Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 26.Papageorgiou A.C. Acharya K.R. Trends Microbiol. 2000; 8: 369-375Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Briefly, it consists of two domains, an N-terminal and a C-terminal one (Fig. 1A). The N-terminal domain comprises a β-barrel known as the oligosaccharide/oligonucleotide binding fold (OB fold). At the top of the barrel, a disulfide bridge is formed by residues Cys-93 and Cys-110 between strands β4 and β5. The loop linking the disulfide bridge is highly flexible, a characteristic feature common among all the staphylococcal and streptococcal superantigens with the exception of TSST-1 and SpeC. This loop cannot be defined with a definite single conformation, and it is most likely able to adopt different conformations. The C-terminal domain has an altered β-grasp motif (a β-sheet packed against an α-helix) structure. In superantigens, this helix is a long central helix (α5) packed diagonally across the molecule. Strands β6, β10, and β12 are parallel to each other and antiparallel to β7 and β9. The N-terminal tail consists of two 310 helices (αN1 and αN2) located on top of the C-terminal domain, and thus it is considered part of this domain. Comparison with Other SEC2 Structures—Superposition of the three crystal forms of SEC2 shows an r.m.s. deviation in Cα atoms of 0.21 and 0.73 for form I-form III and form II-form III, respectively (Table II and Fig. 2A). Residues from the highly flexible disulfide loop were excluded from the calculations because their position is ambiguous. Regions that deviate most include the residues at the N- and C-termini, residues 17-21, 54-58, residues flanking the flexible disulfide loop, and residues 122-130.Table IIComparison of the three crystal forms of SEC2Space groupOrthorhombicTetragonalMonoclinicP212121P43212P21Cell dimensions (Å)42.54 × 44.69 × 131.4243.05 × 43.05 × 290.0043.43 × 69.92 × 42.42β = 90.10°Molecules/asymmetric unit111Resolution (Å)2.02.02.7r.m.s.d.aResidues 96—107 were excluded from the calculationsCα0.210.73Main chain0.220.81All atoms0.551.79No. of water molecules7771No. of zinc ions210a Residues 96—107 were excluded from the calculations Open table in a new tab Zn I Site—This zinc-binding site, site Zn I, is analogous to that found in crystal form I (6.Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure. 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), which is located in a solvent exposed region of the protein at the stretch connecting the N- and C-terminal domains at the bottom of the molecule. In both SEC2 structures the zinc ion is tetrahedrally coordinated to His-118, His-122, Asp-83 from one molecule and Asp-9 (from a symmetry-related molecule in the crystal lattice). The temperature factor for the zinc ion is 13.90 Å2. No zinc site was found in crystal form II, probably because of the removal of the zinc ion after extensive dialysis before crystallization (12.Swaminathan S. Furey W. Pletcher J. Sax M. Nat. Struct. Biol. 1995; 2: 680-686Crossref PubMed Scopus (56) Google Scholar). The r.m.s. deviation of main-chain atoms in this region ranges from 0.33 to 0.62. When compared with the zinc free SEC2 structure, the side-chain atoms of crystal form I Asp-9, Asp-83, His-118, and His-122 have an r.m.s deviation of 0.9-5.6, 0.38-2.1, 0.43-1.0, and 0.56-0.58 Å2, respectively. Zn II Site—The secondary zinc-binding site in SEC2 (Fig. 1B) was found after co-crystallization of SEC2 with 2.5 mm zinc acetate. This site involves two ligands from one molecule (His-47 and Glu-71) and another two from a symmetry-related molecule (Glu-119, Glu-80). For this site to be created, His-47 had to adopt a different conformation by a swing of about 90° from its original position (Fig. 1C). Moreover, Glu-71 had to move ∼2.6 Å away from its original position to accommodate the zinc ion. Glu-119 and Glu-80 both move toward the zinc ion (about 3.1 and 2.0 Å shift from their original position, respectively) to form a ligand for the zinc ion. Structure-based alignment of SEC2 with SEB shows the presence of a phenylalanine residue, Phe-47 (SEB), in a structurally equivalent to that of His-47 (SEC2). This indicates that SEB would be unlikely to form a similar zinc-binding site. No other major conformational changes in the region are observed. The B-factor for the zinc ion is 20.68 Å2, indicating a well ordered metal ion. T cell Activation—T cell activation assays were carried out to establish the effect of zinc on the T cell stimulatory capacity of SEC2. T cell proliferation assays were therefore carried out either in the presence of a range of zinc or magnesium ion concentrations. The results from these experiments (Fig. 3A) demonstrated a complex pattern of T cell response. Firstly, it was clear that SEC2 stimulation of T cells did not absolutely require zinc and that T cells were stimulated by the toxin in its absence. Secondly, at zinc concentrations between 10-100 nm we routinely observed a small but reproducible decrease in responses, however, in the presence of higher concentrations of zinc (up to 100 μm) T cell responses were enhanced ∼1.5-fold on average compared with controls. Most striking, however, was the abolition of cell responses in the presence of high levels of zinc. It was notable that at all parallel doses of magnesium, no effect on T cell responses was observed (Fig. 3B). These T cell responses were clearly MHC class II-dependent, as control CHO transfectants did not stimulate significant T cell responses (data not shown). Overall, these data demonstrate that the effect of zinc on T cell responses to SEC2 was concentration-dependent, and at high doses, highly significant inhibition was observed. Biological Implications—Multiple zinc-binding sites in superantigens have been identified previously (9.Sundstrom M. Abrahmsen L. Antonsson P. Mehindate K. Mourad W. Dohlsten M. EMBO J. 1996; 15: 6832-6840Crossref PubMed Scopus (94) Google Scholar, 13.Li Y. Li H. Dimasi 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 (110) Google Scholar, 27.Li P.L. Tiedemann R.E. Moffat S.L. Fraser J.D. J. Exp. Med. 1997; 186: 375-383Crossref PubMed Scopus (65) Google Scholar). In the case of SpeC, one zinc site is involved in dimerization of the toxin, the other in binding MHC class II molecules. As such, several possible mechanisms for the interaction of this toxin with MHC II and TCR have been proposed (Fig. 2B). The role of the Zn I site in SEC2 has been discussed previously (6.Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure. 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). It was suggested that this site could be used for the binding of a second MHC class II molecule, away from the normal SEB-like MHC-binding site. Binding could take place at both the generic and Zn I sites or exclusively in only one site depending on the conditions. In support of this argument, MHC class II molecules have previously been shown to bind superantigen zinc sites via His-81 from the β-chain of the MHC molecule (13.Li Y. Li H. Dimasi 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 (110) Google Scholar, 14.Petersson K. Hakansson M. Nilsson H. Forsberg G. Svensson L.A. Liljas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (86) Google Scholar). The presentation of some superantigens to T cell receptors is enhanced by the binding of specific peptides (28.Wen R. Cole G.A. Surman S. Blackman M.A. Woodland D.L. J. Exp. Med. 1996; 183: 1083-1092Crossref PubMed Scopus (58) Google Scholar). As the SEB·DR1 crystal structure revealed no contacts between the superantigen and the antigen peptide (in contrast to the TSST-1·DR1 complex), a second MHC-binding site could provide the necessary contacts for the peptide. Interestingly the zinc mediated SpeC·HLA-DR2 (13.Li Y. Li H. Dimasi 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 (110) Google Scholar) and SEH·HLA-DR1 complexes (14.Petersson K. Hakansson M. Nilsson H. Forsberg G. Svensson L.A. Liljas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (86) Google Scholar) show extensive contact (approximately one-third of the contact surface area) between the toxin and the MHC class II-associated antigenic peptide. It has also been suggested that this site may have a structural role, i.e. by providing stability in this region. Nevertheless, the crystal structure of SEC2 without zinc (12.Swaminathan S. Furey W. Pletcher J. Sax M. Nat. Struct. Biol. 1995; 2: 680-686Crossref PubMed Scopus (56) Google Scholar) revealed no major conformational changes in this region. A comparison with the corresponding region in SEB (which does not bind zinc) shows B-factors similar to the average B-factor for the protein (7.Papageorgiou A.C. Tranter H.S. Acharya K.R. J. Mol. Biol. 1998; 277: 61-79Crossref PubMed Scopus (103) Google Scholar) and no major conformational change compared with SEC2. Other possibilities for the role of the Zn I site could also be considered, for example the cross-linking of two SEC2 molecules. This possibility was not discussed previously because of the lack of the TCR·SEC2/SEC3 structures at that time (6.Papageorgiou A.C. Acharya K.R. Shapiro R. Passalacqua E.F. Brehm R.D. Tranter H.S. Structure. 1995; 3: 769-779Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Based on the arrangement of the two symmetry-related SEC2 molecules, binding of a TCR molecule to each of them shows no clashes between the two TCR molecules (Fig. 2C). Moreover, binding of an MHC-class II molecule could also be accommodated to each of the SEC2 molecule. Thus, the cross-linking of two SEC2 molecules leads to the formation of two TCR·SEC2·MHC complexes. However direct experimental data to support this model have yet to be obtained. An SEC2-like zinc-binding site has been found in SEA (29.Schad E.M. Papageorgiou A.C. Svensson L.A. Acharya K.R. J. Mol. Biol. 1997; 269: 270-280Crossref PubMed Scopus (35) Google Scholar) and SED (9.Sundstrom M. Abrahmsen L. Antonsson P. Mehindate K. Mourad W. Dohlsten M. EMBO J. 1996; 15: 6832-6840Crossref PubMed Scopus (94) Google Scholar). These two superantigens, however, possess a primary zinc-binding site at their respective C-terminal domains. Biochemical evidence suggests that its role is either to participate in an MHC-class II molecule binding (SEA) or to create homodimers (SED). The role of the SEC2-like zinc-binding site in SEA and SED is still unknown, but some suggestions have been put forward (29.Schad E.M. Papageorgiou A.C. Svensson L.A. Acharya K.R. J. Mol. Biol. 1997; 269: 270-280Crossref PubMed Scopus (35) Google Scholar) (30.Al-Daccak R. Mehindate K. Damdoumi F. Etongue-Mayer P. Nilsson H. Antonsson P. Sundstrom M. Dohlsten M. Sekaly R.P. Mourad W. J. Immunol. 1998; 160: 225-232PubMed Google Scholar). The Zn II Site—The Zn II site is formed in the presence of zinc and is close to the normal MHC-binding site expected for SEC2 based on the DR1-SEB structure (31.Jardetzky T.S. B" @default.
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• W2003262553 title "Identification of a Secondary Zinc-binding Site in Staphylococcal Enterotoxin C2" @default.
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