Solution and membrane‐bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain

Abstract

During cell intoxication by diphtheria toxin, endosome acidification triggers the translocation of the catalytic (C) domain into the cytoplasm. This event is mediated by the translocation (T) domain of the toxin. Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin. Using partitioning experiments with lipid vesicles, fluorescence spectroscopy, and a lipid vesicle leakage assay, we characterized the dominant behavior of the T domain over the C domain during the successive steps by which these domains interact with a membrane upon acidification: partial unfolding in solution and during membrane binding, and then structural rearrangement during penetration into the membrane. To this end, we compared, for each domain, isolated or linked together in a CT protein (the toxin lacking the receptor‐binding domain), each of these steps. The behavior of the T domain is marginally modified by the presence or absence of the C domain, whereas that of the C domain is greatly affected by the presence of the T domain. All of the steps leading to membrane penetration of the C domain are triggered at higher pH by the T domain, by 0.5–1.6 pH units. The T domain stabilizes the partially folded states of the C domain corresponding to each step of the process. The results unambiguously demonstrate that the T domain acts as a specialized pH‐dependent chaperone for the C domain. Interestingly, this chaperone activity acts on very different states of the protein: in solution, membrane‐bound, and membrane‐inserted.

Diphtheria toxin conformational switching at acidic pH

“…This event is assisted by DT-B, which inserts into the membrane and forms a transmembrane ion channel [6]. Cytosolic chaperones assist the refolding of DT-A on the cytosolic side of the endosomal membrane [4,[7][8][9]. DT-A is then released into the cytosol upon reduction of the interchain disulfide bridge, which is the ratelimiting step of the entire process of cell entry [10].…”

Diphtheria toxin

“…As pH decreases from 7 to 4 (or below), the C domain has also a tendency to partially unfold, adopt a molten, globulelike state, and, as a result, bind and penetrate negatively charged phospholipid bilayers (Figure 4.6) [52,131]. However, these transitions occur at lower pH values and never reach full completion, as compared to those adopted by the T domain.…”

“…The role of the T domain is to assist the C domain in these transitions (Figure 4.6). Under acid conditions, the T domain in solution, as well as bound or inserted into a lipid membrane, has the propensity to bind proteins in a molten globule state [52,131,132]. More precisely, the T domain stabilizes the partially folded states of the C domain corresponding to each step of the process [52].…”

“…Under acid conditions, the T domain in solution, as well as bound or inserted into a lipid membrane, has the propensity to bind proteins in a molten globule state [52,131,132]. More precisely, the T domain stabilizes the partially folded states of the C domain corresponding to each step of the process [52]. These transitions of C (partial unfolding, membrane binding, and membrane penetration) occur at a higher pH of about 1 pH unit than in the absence of T, and they also occur to a fuller extent (Figure 4.6).…”

“…This enables its interaction with the endosomal membrane [45][46][47]. The T domain assists the passage of the C domain across the membrane into the cytosol [48][49][50][51][52]. The disulfide bridge Cys186-Cys201 is reduced and C is released from the rest of the toxin [53,54].…”