Contribution of Subdomain Structure to the Thermal Stability of the Cholera Toxin Al Subunit

    Authors

    T. Banerjee; A. Pande; M. G. Jobling; M. Taylor; S. Massey; R. K. Holmes; S. A. Tatulian;K. Teter

    Comments

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    Abbreviated Journal Title

    Biochemistry

    Keywords

    RETICULUM-ASSOCIATED DEGRADATION; ENDOPLASMIC-RETICULUM; RETRO-TRANSLOCATION; A1 POLYPEPTIDE; PROTEIN; CELLS; CYTOSOL; RICIN; ACTIVATION; ENTRY; Biochemistry & Molecular Biology

    Abstract

    The catalytic Al subunit of cholera toxin (CTAl) is an ADP-ribosyltransferase with three distinct subdomains: CTAl(1) forms the catalytic core of the toxin, CTAl, is an extended linker between CTAl(1) and CTAl(3), and CTAl(3) 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 CTAl(1) subdomain is thermally unstable and that the CTAl(2) subdomain provides a degree of conformational stability to CTAl(1). The CTAl(3) 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 CTAl(3) 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.

    Journal Title

    Biochemistry

    Volume

    49

    Issue/Number

    41

    Publication Date

    1-1-2010

    Document Type

    Article

    Language

    English

    First Page

    8839

    Last Page

    8846

    WOS Identifier

    WOS:000282839900004

    ISSN

    0006-2960

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