Involvement of denaturation-like changes in Pseudomonas exotoxin a hydrophobicity and membrane penetration determined by characterization of pH and thermal transitions: Roles of two distinct conformationally altered states

Previous investigators have shown that exotoxin A undergoes a conformational switch to a hydrophobic state at low pH. This change appears to play a role in exotoxin A entry into cells by facilitating its penetration of the membranes of acidic organelles. We have examined the effects of pH, temperature, and denaturants in order to define the role of conformational changes in membrane penetration by the exotoxin. We find that two distinct low pH conformations exist. An intermediate low pH state (L_I) dominates at pH 3.7-5.4 and is distinguished by blue-shifted fluorescence and weak or no hydrophobicity. The second low pH state (L_II) is dominant below pH 3.7 and is characterized by red shifted fluorescence and strong hydrophobicity. L_I is a folded state as judged by its spectroscopic properties and the observation that it undergoes distinct and cooperative thermal and denaturant induced unfolding transitions. L_II appears to be more like a denatured state, as it shows no cooperative thermal or denaturant induced transitions and has spectroscopic properties very similar to exotoxin A that has been thermally denatured at pH 7. Exotoxin A in the L_II state strongly binds detergent micelles and binds and inserts into model membranes. Therefore, denaturation-like conformational changes appear to play an important role in membrane insertion. The pH of the transition to a membrane-inserting state is influenced by the composition of the model membranes and is close to pH 5 in the presence of vesicles containing a phosphatidylglycerol/phosphatidylcholine mixture. These vesicles probably promote formation of the L_II state via mass action effects. The implications of these results for membrane penetration and translocation of proteins without apparent hydrophobic regions, such as exotoxin A, is discussed.