Effects of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A.
Cysteines 265 and 287 of Pseudomonas aeruginosa exotoxin A (ETA) were substituted by serine, thereby eliminating a disulfide bridge within domain II, the putative membrane insertion-translocation domain. Purified mutant toxin was 80-fold less toxic for mouse L cells than was wild-type ETA while retaining the same specific activity in the ADP-ribosyltransferase reaction as did wild-type toxin. Binding of the nonionic detergent Triton X-114 by mutant ETA occurred at a slightly higher pH than did binding by wild-type ETA, suggesting that the mutant protein more readily undergoes a conformational change exposing hydrophobic regions. Data are presented supporting the notion that the mutant and wild-type toxins enter from the same intracellular compartment. The lower cytotoxicity of the mutant protein could be due to accelerated intracellular degradation or abortive, premature membrane insertion.
Madshus
I H
IH
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Collier
R J
RJ
D036002:ADP Ribose Transferases; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004220:Disulfides; D005098:Exotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009154:Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012694:Serine; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000643:Ammonium Chloride; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D011065:Poly(ADP-ribose) Polymerases; D011994:Recombinant Proteins; D012694:Serine; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A