Isolation of HLA-DR1 center dot(staphylococcal enterotoxin A)(2) trimers in solution

Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules, Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution, Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1 . SEA(2) trimers. The molar ratio of components was determined by dual isotope labeling, The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes, Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.