title: A dipeptide insertion in domain I of exotoxin A that impairs receptor binding.
abstract: Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.
authors.affiliation: Molecular and Cell Biology Program, University of Texas at Dallas, Richardson 75080.
authors.forename: G J
authors.initials: GJ
authors.lastname: Chaudry
authors.forename: R B
authors.initials: RB
authors.lastname: Wilson
authors.forename: R K
authors.initials: RK
authors.lastname: Draper
authors.forename: R C
authors.initials: RC
authors.lastname: Clowes
mesh_terms: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002352:Carrier Proteins; D002872:Chromosome Deletion; D015252:Deoxyribonucleases, Type II Site-Specific; D004151:Dipeptides / Q000235:genetics*; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000235:genetics*; D037521:Virulence Factors; D014961:X-Ray Diffraction; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002352:Carrier Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; D004151:Dipeptides; D005098:Exotoxins; D005971:Glutamates; D018698:Glutamic Acid; D010649:Phenylalanine; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C027553:Pseudomonas exotoxin binding protein, mouse; C027046:TCGA-specific type II deoxyribonucleases