Identification of the Shiga toxin A-subunit residues required for holotoxin assembly.
Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity 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 residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.
Haddad
J E
JE
Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Jackson
M P
MP
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D003062:Codon; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D015183:Restriction Mapping; D017384:Sequence Deletion; D022603:Shiga Toxins; D012760:Shigella / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013728:Terminator Regions, Genetic
D001427:Bacterial Toxins; D003062:Codon; D011994:Recombinant Proteins; D022603:Shiga Toxins