MOLECULAR CHARACTERIZATION OF THE PUTATIVE T-CELL RECEPTOR CAVITY OF THE SUPERANTIGEN STAPHYLOCOCCAL-ENTEROTOXIN-B

A number of investigators have utilized a variety of methods to identi fy the structural basis for the interaction of superantigens with the T-cell receptor beta-chain. The previous studies strongly suggest that a region of the toxin near residues N23, Y61, Y91 and D209 is importa nt for this binding activity. Examination of crystal structure data sh ows that these residues line the rim of one side of a shallow cavity i n the toxin. In an attempt further to define the face of the staphyloc occal enterotoxin B (SEB) molecule involved in the interaction with th e beta-chain, we have employed a polymerase chain reaction (PCR)-based , site-specific mutagenesis method to generate amino acid substitution s of residues on the opposite side of this putative T-cell receptor in teraction cavity. Our results show that Y175 and N179 appear to be inv olved in the function of this superantigen, since each of several subs titutions at this position exhibits a significantly reduced ability to induce T-cell proliferation. At the same time, mutation of the proxim al Y186 does not alter the superantigen activity of SEE. Binding analy sis of these mutants shows that class II binding activity is not signi ficantly altered. Analysis of the responding T cells shows that the mu tant toxins maintain T-cell receptor V beta selectivity. However, resp onses of T cells bearing the V beta 8.1 allele appear to be particular ly diminished. When viewed in the context of other results reported in the literature, our results suggest that the T-cell receptor interact ion site involves SEE residues which ring both the Y175/N179-side and the N23-side of a cavity on one side of the toxin molecule.