IDENTIFICATION OF THE SHIGA TOXIN A-SUBUNIT RESIDUES REQUIRED FOR HOLOTOXIN ASSEMBLY

Recent X-ray crystallographic analyses have demonstrated that the rece ptor-binding (B) subunits of Shiga toxin (STX) are arranged as a dough nut-shaped pentamer. The C terminus of the enzymatic (A) subunit presu mably penetrates the nonpolar pore of the STX B pentamer, and the holo toxin is stabilized by noncovalent interactions between the polypeptid es. We identified a stretch of nine nonpolar amino acids near the C te rminus of StxA which were required for subunit association by using si te-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor ana log enzyme-linked immunosorbent assay, immunoprecipitation, and a cyto toxicity assay. Tryptophan and aspartic acid residues which form the N -terminal boundary, as well as two arginine residues which form the C- terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residu es 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore w hile the tryptophan, aspartic acid, and 2 arginine residues interact w ith other charged or aromatic amino acids outside the pore on the plan ar surfaces of the STX B pentamer.