A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

Pseudomonas exotoxin A (PE) is a cytotoxin composed of three structural dom ains, Domain I is responsible for cell binding, domain II for membrane tran slocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death, To investig ate the role of the six alpha-helices (A-F) that form the translocation dom ain, we deleted them successively one at a time, All mutants showed native cell-binding and catalytic activities, indicating that deletions specifical ly affected translocation activity. This step of the intoxication procedure was examined directly using a cell-free translocation assay, and indirectl y by monitoring cytotoxicity. Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limi ting for PE intoxication, Deletion of B, C and D helices resulted in non-to xic and non-translocating molecules, whereas mutants lacking the A or E hel ix displayed significant cytotoxicity albeit 500-fold lower than native PE. We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparative ly dispensable. The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% high er than PE, along with a three- to sixfold increase in cytotoxicity in all tested cell lines. This toxin is the most in vitro active PE mutant obtaine d until now. Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active mol ecule.