Abstract
The catalytic Al subunit of cholera toxin (CTAl) is an ADP-ribosyltransferase with three distinct subdomains: CTAl1 forms the catalytic core of the toxin, CTAl, is an extended linker between CTAl1 and CTAl3, and CTAl3 is a compact globular region. CTAl crosses the endoplasmic reticulum (ER) membrane to enter the cytosol where it initiates a cytopathic effect. Toxin translocation involves ER-associated degradation (ERAD), a quality control system that exports misfolded proteins from the ER to the cytosol. At the physiological temperature of 37 degrees C, the free CTAl subunit is in a partially unfolded conformation that triggers its ERAD-mediated translocation to the cytosol. Thus, the temperature sensitivity of CTAl structure is an important determinant of its function. Here, we examined the contribution of CTAl subdomain structure to the thermal unfolding of CTAl. Biophysical measurements demonstrated that the CTAl1 subdomain is thermally unstable and that the CTAl2 subdomain provides a degree of conformational stability to CTAl1. The CTAl3 subdomain does not affect the overall stability of CTAl, but the thermal unfolding of CTAl appears to begin with a local loss of structure in the CTAl3 subdomain: glycerol and acidic pH both inhibited the thermal disordering of full-length CTAl but not the disordering of a CTAl construct lacking the Al-3 subdomain. These observations provide mechanistic insight regarding the thermal unfolding of CTAl, an event which facilitates its subsequent translocation to the cytosol.