The relationship of structure to function of the superantigen Staphylococcal enterotoxin A
The relationship of structure to function in the superantigens of Staphylococcus aureus
1 Enterotoxins were isolated from expression in E. coli using the pGeX vector. Yields of up to 20 mg/litre of culture of purified toxin were achieved.
2 Recombinant SEA, SEB, SED and SEE produced by this system were identical to the wild type enterotoxins by several biological and biochemical criteria.
3 The unique T-cell receptor binding site of SEA and SEE was identified by the use of a panel of hybrids between these two toxins. Four different assays showed that the different V? specificity of these toxins was determined by G200, S206 and N207 in SEA and at P206 and D207 in SEE.
4 Residues on both SEA and HLA-DR were identified that directly interact upon binding, thus defining the MHC class II binding site of SEA. The requirement of zinc for SEA binding to HLA-DR allowed the identification of the MHC class II binding site on SEA. This requirement was shown to be due to zinc bridging between these proteins. The zinc binding residues in SEA were demonstrated to be H187, H225 and D227.
5 Histidine 81 in the ?-chain of HLA-DR1 was shown to be the fourth residue in the tetravalent coordination of zinc.
6 This investigation in conjunction with the crystal structure of SEB and other mutational studies has collectively identified the T-cell receptor binding site of the enterotoxins. This study also located the MHC class II binding site of SEA and on the crystal structure of SEB the zinc coordinating residues cluster together, well separated from the T-cell receptor binding site. Thus proving that these superantigens cross link the T-cell receptor and MHC class II antigen.
7 The location of the zinc coordinating residues together with the crystal structures of SEB and HLA-DR1 allowed the construction of a structural model of how SEA binds to HLA-DR1. This model predicts that SEA binds across the end of the peptide groove and significantly that the orientation of the T-cell receptor in the recognition of the SEA-MHC class II complex is different to the orientation used to recognise peptide-MHC. This non conventional recognition of superantigen is compatible with the influence of polymorphism in both the T-cell receptor and MHC class II on superantigen recognition.