Low pH-induced changes in Pseudomonas exotoxin and its domains: increased binding of Triton X-114.
Pseudomonas exotoxin (PE), which is composed of three structural domains, is a 66-kilodalton protein secreted by P. aeruginosa that is cytotoxic for mammalian cells. After binding to cell surface receptors and internalization into low-pH endocytic vesicles, PE or an active fragment kills mammalian cells by translocating across an intracellular membrane to the cytoplasm and shutting down protein synthesis. To investigate possible conformational changes associated with the translocation process, full-length PE or recombinant proteins containing the PE cell recognition domain, translocation domain, enzymatic domain, or translocation plus enzymatic domains were incubated with Triton X-114 at pH values ranging from 3.0 to 7.0. The truncated forms used were intact domains that had been expressed in Escherichia coli and subsequently purified. Previous studies (K. Sandvig and J. O. Moskaug, Biochem. J. 245:899-901, 1987) had shown that full-length PE bound more Triton X-114 at a low pH than at a physiologic pH. Therefore, we investigated whether this increased binding was due to a global change in PE or a change within a particular domain. Results showed that all the truncated toxin proteins displayed a similar pH-dependent entry into the detergent phase as native PE, with a transition point of 4.2 for PE and 4.4 to 4.5 for the truncated toxins. The isoelectric points of the recombinant proteins were measured and indicate that, at a low pH (5.0), the cell recognition domain bears a net positive charge, the translocation domain bears a net negative charge, and the enzymatic domain bears no charge. The results suggest that upon acidification in the endosome, PE becomes globally hydrophobic and is converted into a translocation-competent form.
Idziorek
T
T
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
FitzGerald
D
D
Pastan
I
I
D036002:ADP Ribose Transferases; D001426:Bacterial Proteins / Q000235:genetics / Q000648:ultrastructure; D001427:Bacterial Toxins; D001692:Biological Transport; D003001:Cloning, Molecular; D005098:Exotoxins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D010446:Peptide Fragments / Q000378:metabolism; D011092:Polyethylene Glycols / Q000378:metabolism; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000472:pathogenicity*; D011994:Recombinant Proteins; D012995:Solubility; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005098:Exotoxins; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A