Folding Changes in Membrane-Inserted Diphtheria Toxin That May Play Important Roles in Its Translocation

Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L′ predominates below 25–30 °C, and the other, L″, predominates above 25–30 °C and is formed from the L′ state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L′ and L″ conformations give rise to two new conformations, R′ and R″, respectively. The R′ and R″ conformations differ from each other in that in the R′ state the A domain remains folded, whereas in the R″ state the A domain is unfolded. This is confirmed by the finding that only the R′ state possesses the capacity to bind and hydrolyze NAD⁺. It is also supported by the finding that the R″ state can also be formed by thermal unfolding of the R′ state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer. Since the two different R states arise from the two different L states, it is likely that the L′ state contains folded A domain and the L″ conformation contains unfolded A domain. It has also been found that after pH neutralization the release of the A domain by disulfide reduction of membrane-inserted toxin can result in A domain refolding. On the basis of this result and the finding that the less folded L″ and R″ forms of the toxin predominate at 37 °C, we conclude that the behavior of the toxin at physiological temperatures is consistent with translocation models proposing an unfolding/refolding cycle.