title: Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis.
abstract: Shiga toxin (STX), a bacterial toxin produced by Shigella dysenteriae type 1, is a hexamer composed of five receptor-binding B subunits which encircle an alpha-helix at the carboxyl terminus of the enzymatic A polypeptide. Hybrid toxins constructed by fusing the A polypeptide sequences of STX and Shiga-like toxin type II were used to confirm that the carboxyl terminus of the A subunits governs association with the B pentamers. The alpha-helix of the 293-amino-acid STX A subunit contains nine residues (serine 279 to methionine 287) which penetrate the nonpolar pore of the B-subunit pentamer. Site-directed mutagenesis was used to establish the involvement of two residues bordering this alpha-helix, aspartic acid 278 and arginine 288, in coupling the C terminus of StxA to the B pentamer. Amino acid substitutions at StxB residues arginine 33 and tryptophan 34, which are on the membrane-contacting surface of the pentamer, reduced cytotoxicity without affecting holotoxin formation. Although these B-subunit mutations did not involve receptor-binding residues, they may have induced an electrostatic repulsion between the holotoxin and the mammalian cell membrane or disrupted cytoplasmic translocation.
authors.affiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: C
authors.initials: C
authors.lastname: Jemal
authors.forename: J E
authors.initials: JE
authors.lastname: Haddad
authors.forename: D
authors.initials: D
authors.lastname: Begum
authors.forename: M P
authors.initials: MP
authors.lastname: Jackson
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins; D022603:Shiga Toxins; D012762:Shigella dysenteriae / Q000472:pathogenicity; D013329:Structure-Activity Relationship
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D011994:Recombinant Proteins; D022603:Shiga Toxins