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.
Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (H N ) and a receptor-binding domain (H C ). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by H N . We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and H N of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and H N increases as pH drops, and that H N further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of H N . This property is downplayed when LC is linked to H N . H N thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.
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