Upon entering mammalian cells, Pseudomonas exotoxin A (PE) is proteolytical
ly processed by furin to produce an N-terminal fragment of 28 kDa and a C-t
erminal fragment of 37 kDa. Cleavage is followed by the reduction of a key
disulfide bond (cysteines 265-287). This combination of proteolysis and red
uction releases the 37 kDa C-terminal fragment, which then translocates to
the cytosol where it ADP-ribosylates elongation factor 2 and inhibits prote
in synthesis. To investigate toxin reduction, furin-nicked PE or a hypercle
avable mutant, PEW281A, was subjected to various treatments and then analyz
ed for fragment production. Reduction was evident only when unfolding condi
tions and a reducing agent were applied. Thermal unfolding of PE, as eviden
ced by changes in or-helical content and increased sensitivity to trypsin,
rendered nicked toxin susceptible to protein disulfide isomerase- (PDI-) me
diated reduction. When subcellular fractions from toxin-sensitive cells wer
e incubated with nicked PE, toxin unfolding and reducing activities were pr
esent in the membrane fraction but not the soluble fraction. These data ind
icate that PE reduction is a two-step process: unfolding that allows access
to the Cys265-287 disulfide bond, followed by reduction of the sulfur-sulf
ur bond by PDI or a PDI-like enzyme. With regard to cellular processing, we
propose that the toxin's three-dimensional structure retains a "closed" co
nformation that restricts solvent access to the Cys265-287 disulfide bond u
ntil after a cell-mediated unfolding event.