The Co-crystal Structure of Staphylococcal Enterotoxin Type A With Zn2+ at 2.7 Å Resolution

Superantigens form complexes with major histocompatibility complex (MHC) class II molecules and T-cell receptors resulting in extremely strong immunostimulatory properties. Staphylococcus aureus enterotoxin A (SEA) belongs to a subgroup of the staphylococcal superantigens that utilizes Zn2+ in the high affinity interaction with MHC class II molecules. A high affinity metal binding site was described previously in SEA co-crystallized with Cd2+ in which the metal ion was octahedrally co-ordinated, involving the N-terminal serine. We have now co-crystallized SEA with its native co-factor Zn2+ and determined its crystal structure at 2.7 Å resolution. As expected for a Zn2+ ion, the co-ordination was found to be tetrahedral. Three of the ligands are located on the SEA surface on a C-terminal domain β-sheet, while the fourth varies with the conditions. Further analysis of the zinc binding event was performed using titration microcalorimetry, which showed that SEA binds Zn2+ with an affinity of KD = 0.3 μM in an entropy driven process. The differential Zn2+ co-ordination observed here has implications for the mechanism of the SEA-MHC class II interaction. Superantigens form complexes with major histocompatibility complex (MHC) class II molecules and T-cell receptors resulting in extremely strong immunostimulatory properties. Staphylococcus aureus enterotoxin A (SEA) belongs to a subgroup of the staphylococcal superantigens that utilizes Zn2+ in the high affinity interaction with MHC class II molecules. A high affinity metal binding site was described previously in SEA co-crystallized with Cd2+ in which the metal ion was octahedrally co-ordinated, involving the N-terminal serine. We have now co-crystallized SEA with its native co-factor Zn2+ and determined its crystal structure at 2.7 Å resolution. As expected for a Zn2+ ion, the co-ordination was found to be tetrahedral. Three of the ligands are located on the SEA surface on a C-terminal domain β-sheet, while the fourth varies with the conditions. Further analysis of the zinc binding event was performed using titration microcalorimetry, which showed that SEA binds Zn2+ with an affinity of KD = 0.3 μM in an entropy driven process. The differential Zn2+ co-ordination observed here has implications for the mechanism of the SEA-MHC class II interaction. INTRODUCTIONSuperantigens bind as nonprocessed proteins to major histocompatibility (MHC) 1The abbreviations used are: MHCmajor histocompatibility complexSEstaphylococcal enterotoxin(s)MES4-morpholineethanesulfonic acid. class II molecules on antigen presenting cells and subsequently activate T-lymphocytes by interactions with T-cell receptors. Superantigen activated T-cells proliferate vigorously, and subsequently T-cell and monocyte derived cytokines are produced in large amounts. The released cytokines contribute to the development of toxin-induced disease processes (for a review see 1Scherer M.T. Ignatowicz L. Winslow G.M. Annu. Rev. Cell Biol. 1993; 9: 101-128Crossref PubMed Scopus (220) Google Scholar).The best characterized superantigens are the staphylococcal enterotoxins. Based on sequence similarity, these may be divided into two subgroups: the first consists of staphylococcal enterotoxins A, D, E, and H (SEA SED, SEE, and SEH) and the second of staphylococcal enterotoxins B and C1-C3 (SEB, SEC1, SEC2, and SEC3) (reviewed in 2Marrack P. Kappler J. Science. 1990; 248: 705-711Crossref PubMed Scopus (1213) Google Scholar). The sequence identity of SEA to other staphylococcal enterotoxins ranges from 25 (SEC1) to 83% (SEE). In addition, SEA, SED, and SEE are all dependent on Zn2+ for high affinity binding to MHC class II molecules in contrast to SEB and SEC1-3 that bind MHC class II molecules independently of metal ions (3Fraser J.D. Urban R.G. Strominger J.L. Robinson H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5507-5511Crossref PubMed Scopus (113) Google Scholar).Recently solved crystal structures of the free forms of SEA (4Schad 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), SEB (5Swaminathan S. Furey W. Pletcher J. Sax M. Nature. 1992; 359: 801-806Crossref PubMed Scopus (280) Google Scholar), SEC2 (6Papageorgiou 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 (111) Google Scholar), and toxic shock syndrome toxin 1 (7Acharya 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, 8Prasad G.S. Earhart C.A. Murray D.L. Novick R.P. Schlievert P.M. Ohlendorf D.H. Biochemistry. 1993; 32: 13761-13766Crossref PubMed Scopus (113) Google Scholar), as well as of the SEB-MHC class II complex (9Jardetzky T.S. Brown J.H. Gorga J.C. Stern L.J. Urban R.G. Chi Y. Stauffacher C. Strominger J.L. Wiley D.C. Nature. 1994; 368: 711-718Crossref PubMed Scopus (500) Google Scholar), have created an understanding for the structural constraints by which superantigens interact with their target receptors. The structure of SEB, bound to a MHC class II molecule, confirmed that the superantigen binds to the α-chain of the MHC class II molecule, outside the peptide antigen-binding groove. The more distantly related superantigen, toxic shock syndrome toxin 1, binds in a fashion similar to that of SEB, although it covers a larger area on the receptor and in addition utilizes a bound peptide antigen in the interactions (10Kim J. Urban R.G. Strominger J.L. Wiley D.C. Science. 1994; 266: 1870-1874Crossref PubMed Scopus (247) Google Scholar).Site-directed mutagenesis of SEA confirmed that co-ordination of Zn2+ is required for high affinity binding to MHC class II molecules. It was also shown that SEA most likely binds bivalently to both the α- and the β-chain, of two separate MHC class II molecules, utilizing a surface corresponding to the site previously defined in SEB in the first case and the Zn2+ binding site in the latter (11Abrahmsén L. Dohlsten M. Segren S. Bjork P. Jonsson E. Kalland T. EMBO J. 1995; 14: 2978-2986Crossref PubMed Scopus (147) Google Scholar). SEB in contrast, binds monovalently to only the α-chain.The recently determined crystal structures of the free forms of SEA co-crystallized with Cd2+ (SEA-Cd2+), and SEC2 revealed a metal binding site in each protein. An octahedrally co-ordinated Cd2+ ion in SEA was located on the surface of the β-sheet of the C-terminal domain, whereas a tetrahedral co-ordination of a Zn2+ ion in SEC2 is observed at the interface between the N- and the C-terminal domains.In this study, the crystal structure of SEA, co-crystallized with its native "co-factor" Zn2+ at 2.7 Å resolution is presented and compared with the previously described SEA-Cd2+ structure. Further, the Zn2+ binding is analyzed using titration microcalorimetry. The biological implications of the mode of Zn2+ co-ordination in SEA are discussed with emphasis on metal ion assisted SEA-MHC class II interactions.DISCUSSIONZinc ions are essential for the activity of many enzymes and a structural component in many protein-DNA and protein-protein interactions. A requirement of Zn2+ to obtain the strong affinity between the SEA and MHC class II molecules was first shown by Fraser and co-workers (3Fraser J.D. Urban R.G. Strominger J.L. Robinson H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5507-5511Crossref PubMed Scopus (113) Google Scholar). The amino acid residues involved in the co-ordination of a Zn2+ ion on the SEA surface were identified by site-directed mutagenesis (11Abrahmsén L. Dohlsten M. Segren S. Bjork P. Jonsson E. Kalland T. EMBO J. 1995; 14: 2978-2986Crossref PubMed Scopus (147) Google Scholar, 12Fraser J.D. Lowe S. Irwin M.J. Gascoigne N.R.J. Hudson K.R. Huber B.T. Palmer E. Superantigens: A Pathogens View of the Immune System. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 7-29Google Scholar). When the residues Phe47, Asn128, His187, His225, or Asp227 are substituted for alanines the ability to induce MHC class II-dependent T-cell proliferation is markedly reduced (11Abrahmsén L. Dohlsten M. Segren S. Bjork P. Jonsson E. Kalland T. EMBO J. 1995; 14: 2978-2986Crossref PubMed Scopus (147) Google Scholar). Because histidines and aspartates are preferred zinc ligands, it was speculated that the lowered bioactivity was due to impaired zinc binding and metal ion-dependent SEA-MHC class II interactions for His187, His225, and Asp227 (and possibly also for Asn128). A disruption of a SEB-like interaction with the MHC class II molecule α-chain was expected for the Phe47 to alanine substitution. As shown in the SEA-Cd2+ structure (4Schad 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), the effects of the substitutions described above could be explained with disruption of the metal binding site. His187, His225, and Asp227 were shown to be direct high affinity zinc ligands, and Asn128 was shown to stabilize the conformation of Asp227 via a strong hydrogen bond. However, a comparison between the SEA-Cd2+ structure and the SEA-Zn2+ structure presented here reveals that the two refined structures are virtually identical, but with one important exception: the co-ordination of the bound metal ion.In the present structure the metal ion is co-ordinated without involvement of the N-terminal serine. We clearly observe a tetrahedral Zn2+ co-ordination in both molecules of the asymmetric unit. The two independent molecules in the crystal asymmetric unit have their respective metal binding site in different environments. In both molecules the three high affinity Zn2+ ligands are His187, His225, and Asp227. The fourth ligand, however, is His61 of the neighboring molecule in one case and a water molecule in the second. Thus, none of the molecules of the asymmetric unit utilizes the N terminus in Zn2+ co-ordination as observed for SEA co-crystallized with Cd2+. In fact, the N terminus (residues 1-9) is unordered in each of the molecules.If a Zn2+ ion could be octahedrally co-ordinated as the Cd2+ ion in the SEA-Cd2+ structure, one would expect this situation also in this SEA-Zn2+ structure, at least in the second molecule where no symmetry molecule interactions are observed, instead a water molecule is used as the fourth ligand in tetrahedral co-ordination. However, one possible explanation to this contradiction could be due to different crystallization conditions. Although both Zn2+ and Cd2+ ions could adopt tetrahedral as well as octahedral co-ordination in nonprotein environments, the norm for protein bound Zn2+ ions is tetrahedral (reviewed in 13Valleé B.L. Auld D.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 220-224Crossref PubMed Scopus (390) Google Scholar).The thermodynamic properties for the SEA-Zn2+ 1:1 complex are dominated by the large and positive entropy, ΔSo = 146 J (K mol)−1. The possibility to perform a rigorous thermodynamic analysis of the process in terms of dissecting the different contributions to the thermodynamic properties is dependent on heat capacity data, which at the moment are not available. However, the most likely dominating contribution to positive entropy change are ΔShydr-prot, which is the change in hydration of the protein when Zn2+ is bound, and ΔShydr-Zn, which is the change in entropy for transferring the fully hydrated Zn2+ ion to the protein binding site. In addition, either positive or negative contributions from ΔSion, the entropy change upon proton exchange when binding a ligand to a protein, can occur. The sign of this latter contribution depends on whether there is a positive or a negative proton linkage in the reaction.One possible interpretation of these thermodynamic properties is that a reduction in conformational degrees of freedom occurs upon metal binding, subsequently dehydrating hydrophobic surface residues. An ordering of the N terminus upon Zn2+ binding could possibly explain the thermodynamic properties discussed above, although not observed in any of the two molecules in the asymmetric unit of the present crystal structure. In this context it should be stressed that the form of SEA used here is the product from predicted signal peptide processing, whereas SEA purified from its native host Staphylococcus aureus is a mixture of this and of two truncated forms lacking three or five N-terminal residues, with the latter two as the major forms.2 Thus, none of the shorter forms could co-ordinate the metal ion as observed in the SEA-Cd2+ structure. A thermodynamic analysis of such truncated variants of SEA would be invaluable to the interpretation of the biological significance of the differential metal ion binding modes observed in the SEA-Cd2+ and the present structure.The crystal structure of SEC2 revealed a bound zinc ion in the domain interface region, a metal binding site distinct from the one observed in SEA. The Zn2+ co-ordinating residues were Asp83, His118, and His122 from one molecule and Asp9 from a neighboring molecule in the crystal lattice (6Papageorgiou 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 (111) Google Scholar). In contrast to SEA, SED and SEE, SEB/SEC-MHC class II molecule interactions do not require zinc ions. Thus, the most likely explanation for the function of the zinc ion bound to SEC2 is that it serves a structural function, possibly by stabilizing the domain-domain interactions. However, from a crystallographical point of view, this situation resembles the case for SEA-Zn2+ observed in this study. As described above, one of the molecules in the asymmetric unit utilizes His61 of the neighboring molecule as the fourth Zn2+ ligand. Thus the loop 59-63, that normally is highly mobile, becomes ordered due to the zinc-mediated protein-protein interaction.Interestingly, in the formation of the SEA-MHC class II complex, three Zn2+ ligands are postulated to be derived from the superantigen and the fourth from the receptor (11Abrahmsén L. Dohlsten M. Segren S. Bjork P. Jonsson E. Kalland T. EMBO J. 1995; 14: 2978-2986Crossref PubMed Scopus (147) Google Scholar). The postulated co-ordinating residue in the MHC class II β-chain is an exposed histidine residue, His81 (14Herman A. Labrecque N. Thibodeau J. Marrack P. Kappler J.W. Sekaly R.-P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9954-9958Crossref PubMed Scopus (120) Google Scholar). Thus, the N terminus as oriented in the SEA-Cd2+ structure would have to disengage from the metal ion in order to allow the ligand function of the MHC class II residue. The ligand function of His61 from the neighboring SEA molecule in the asymmetric unit observed in the current structure may thus mimic the SEA-MHC class II β-chain Zn2+-dependent interaction. Judged from the previous SEA-Cd2+ and the present SEA-Zn2+ co-crystal structure, a model for this interaction could be regarded in which the N terminus of SEA can be utilized in the co-ordination of zinc but is released upon MHC class II molecule interactions. A second option would be that the N terminus is not at all involved in zinc binding. The latter case will most surely exist in vivo where naturally occurring SEA can have three or five residues removed in the N terminus compared with the material used in this study and by Schad and co-workers (4Schad 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). However, a full understanding of the interactions in the SEA-MHC class II molecule complex will have to await the crystal structure of such a protein complex. INTRODUCTIONSuperantigens bind as nonprocessed proteins to major histocompatibility (MHC) 1The abbreviations used are: MHCmajor histocompatibility complexSEstaphylococcal enterotoxin(s)MES4-morpholineethanesulfonic acid. class II molecules on antigen presenting cells and subsequently activate T-lymphocytes by interactions with T-cell receptors. Superantigen activated T-cells proliferate vigorously, and subsequently T-cell and monocyte derived cytokines are produced in large amounts. The released cytokines contribute to the development of toxin-induced disease processes (for a review see 1Scherer M.T. Ignatowicz L. Winslow G.M. Annu. Rev. Cell Biol. 1993; 9: 101-128Crossref PubMed Scopus (220) Google Scholar).The best characterized superantigens are the staphylococcal enterotoxins. Based on sequence similarity, these may be divided into two subgroups: the first consists of staphylococcal enterotoxins A, D, E, and H (SEA SED, SEE, and SEH) and the second of staphylococcal enterotoxins B and C1-C3 (SEB, SEC1, SEC2, and SEC3) (reviewed in 2Marrack P. Kappler J. Science. 1990; 248: 705-711Crossref PubMed Scopus (1213) Google Scholar). The sequence identity of SEA to other staphylococcal enterotoxins ranges from 25 (SEC1) to 83% (SEE). In addition, SEA, SED, and SEE are all dependent on Zn2+ for high affinity binding to MHC class II molecules in contrast to SEB and SEC1-3 that bind MHC class II molecules independently of metal ions (3Fraser J.D. Urban R.G. Strominger J.L. Robinson H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5507-5511Crossref PubMed Scopus (113) Google Scholar).Recently solved crystal structures of the free forms of SEA (4Schad 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), SEB (5Swaminathan S. Furey W. Pletcher J. Sax M. Nature. 1992; 359: 801-806Crossref PubMed Scopus (280) Google Scholar), SEC2 (6Papageorgiou 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 (111) Google Scholar), and toxic shock syndrome toxin 1 (7Acharya 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, 8Prasad G.S. Earhart C.A. Murray D.L. Novick R.P. Schlievert P.M. Ohlendorf D.H. Biochemistry. 1993; 32: 13761-13766Crossref PubMed Scopus (113) Google Scholar), as well as of the SEB-MHC class II complex (9Jardetzky T.S. Brown J.H. Gorga J.C. Stern L.J. Urban R.G. Chi Y. Stauffacher C. Strominger J.L. Wiley D.C. Nature. 1994; 368: 711-718Crossref PubMed Scopus (500) Google Scholar), have created an understanding for the structural constraints by which superantigens interact with their target receptors. The structure of SEB, bound to a MHC class II molecule, confirmed that the superantigen binds to the α-chain of the MHC class II molecule, outside the peptide antigen-binding groove. The more distantly related superantigen, toxic shock syndrome toxin 1, binds in a fashion similar to that of SEB, although it covers a larger area on the receptor and in addition utilizes a bound peptide antigen in the interactions (10Kim J. Urban R.G. Strominger J.L. Wiley D.C. Science. 1994; 266: 1870-1874Crossref PubMed Scopus (247) Google Scholar).Site-directed mutagenesis of SEA confirmed that co-ordination of Zn2+ is required for high affinity binding to MHC class II molecules. It was also shown that SEA most likely binds bivalently to both the α- and the β-chain, of two separate MHC class II molecules, utilizing a surface corresponding to the site previously defined in SEB in the first case and the Zn2+ binding site in the latter (11Abrahmsén L. Dohlsten M. Segren S. Bjork P. Jonsson E. Kalland T. EMBO J. 1995; 14: 2978-2986Crossref PubMed Scopus (147) Google Scholar). SEB in contrast, binds monovalently to only the α-chain.The recently determined crystal structures of the free forms of SEA co-crystallized with Cd2+ (SEA-Cd2+), and SEC2 revealed a metal binding site in each protein. An octahedrally co-ordinated Cd2+ ion in SEA was located on the surface of the β-sheet of the C-terminal domain, whereas a tetrahedral co-ordination of a Zn2+ ion in SEC2 is observed at the interface between the N- and the C-terminal domains.In this study, the crystal structure of SEA, co-crystallized with its native "co-factor" Zn2+ at 2.7 Å resolution is presented and compared with the previously described SEA-Cd2+ structure. Further, the Zn2+ binding is analyzed using titration microcalorimetry. The biological implications of the mode of Zn2+ co-ordination in SEA are discussed with emphasis on metal ion assisted SEA-MHC class II interactions.