title: Contribution of subdomain structure to the thermal stability of the cholera toxin A1 subunit.
abstract: The catalytic A1 subunit of cholera toxin (CTA1) is an ADP-ribosyltransferase with three distinct subdomains: CTA1(1) forms the catalytic core of the toxin, CTA1(2) is an extended linker between CTA1(1) and CTA1(3), and CTA1(3) is a compact globular region. CTA1 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 °C, the free CTA1 subunit is in a partially unfolded conformation that triggers its ERAD-mediated translocation to the cytosol. Thus, the temperature sensitivity of CTA1 structure is an important determinant of its function. Here, we examined the contribution of CTA1 subdomain structure to the thermal unfolding of CTA1. Biophysical measurements demonstrated that the CTA1(1) subdomain is thermally unstable and that the CTA1(2) subdomain provides a degree of conformational stability to CTA1(1). The CTA1(3) subdomain does not affect the overall stability of CTA1, but the thermal unfolding of CTA1 appears to begin with a local loss of structure in the CTA1(3) subdomain: glycerol and acidic pH both inhibited the thermal disordering of full-length CTA1 but not the disordering of a CTA1 construct lacking the A1(3) subdomain. These observations provide mechanistic insight regarding the thermal unfolding of CTA1, an event which facilitates its subsequent translocation to the cytosol.
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32826, United States.
authors.forename: Tuhina
authors.initials: T
authors.lastname: Banerjee
authors.forename: Abhay
authors.initials: A
authors.lastname: Pande
authors.forename: Michael G
authors.initials: MG
authors.lastname: Jobling
authors.forename: Michael
authors.initials: M
authors.lastname: Taylor
authors.forename: Shane
authors.initials: S
authors.lastname: Massey
authors.forename: Randall K
authors.initials: RK
authors.lastname: Holmes
authors.forename: Suren A
authors.initials: SA
authors.lastname: Tatulian
authors.forename: Ken
authors.initials: K
authors.lastname: Teter
mesh_terms: D002772:Cholera Toxin / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D002772:Cholera Toxin