Abstract:
The superantigen staphylococcal enterotoxin A (SEA) binds MHC class II molecules via two distinct sites located on opposite sides of each molecule, C-terminal residues of SEA mediate high-affinity binding to pH81 of HLA-DR through a coordinated zinc atom (Hudson et al., 1995). An alternative low-affinity binding site was identified in the N-terminal domain of SEA and was shown to be analogous to the SEB site for the al domain of DR. The two binding sites do not overlap, making possible the formation of MHCi1SEA2 trimers. The existence of these was first suggested by competition studies. Subsequently HLA-DR11.SEA2 complexes were isolated in solution, demonstrating that two SEA molecules can bind a single class II molecule. Binding oftwo SEA molecules to one DR1 molecule is cooperative in both directions. However because the SEA/DRip site is of greater affinity than the SEA/DRla interaction, cooperativity is most important for binding of SEA to the DR? chain. Thus binding of SEA is at least a two-step process initiated by high-affinity binding to the DRp chain. A second SEA molecule is then co-opted to the DR? chain site. As well as activating T cells, SEA activates antigen-presenting cells (APC) by cross-linking MHC class II molecules. Studies with mutant forms of SEA indicate that a single SEA molecule must simultaneously bind two MHC class II molecules at the APC surface for superantigen activity. Oligomerisation of the MHC receptor by SEA results in intracellular signalling in APCs and is essential for the activation of B cells, monocytes and T cells. A structural model of SEA bound to HLA-DR1 is presented. The model predicts that: SEA bridges between DR1 molecules to activate APC; that cooperativity results from an interface between two SEA molecules coligated to the same DR1; and that the nature of TCR ligation by SEA may differ from that of SEB. The data presented in this thesis support a unifying model in which the bacterial superantigens utilise a common structural motif for binding to the a chain of MHC class II which acts as a 'generic' site. Residues F47.L48 are central to the motifin SEA, corresponding to F44.L45 in SEB. Similar residues are conserved in SEC1-3, SED, SEE & SPE-A. One toxin which appears to lack a binding site for the generic MHC class II a-chain site is streptococcal pyrogenic exotoxin C (SPE-C). However this superantigen, like SEA, activates APC. A homodimer form of SPE-C was isolated suggesting this toxin employs a novel method to cross-link MHC class II molecules, distinct from that of SEA, in which the active molecule is a dimer with two zinc-containing p-chain binding sites.