Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1

Abstract

The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature. In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

“…S5). A similar observation was previously made by Goins and Friere with the technique of differential scanning calorimetry (37). Thus, PDI apparently removes CTA1 from CTA2/CTB 5 by a mechanism that does not involve unfolding of the holotoxin-associated CTA1 subunit.…”

“…It thus appeared that PDI can interact with the folded conformations of CTA1 present at low temperatures but not with the unfolded CTA1 conformations present at physiological or near-physiological temperatures. Thermal instability in the CTA1 polypeptide is only apparent after dissociation from the holotoxin (17,37,38), which explains why reduced PDI could interact with CTA1 when it was present in the CT holotoxin at 37°C (Fig. 1B).…”

Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding

“…In contrast, the A-and Bsubunits of cholera toxin unfold independently. This observation was confirmed by the fact that the T m and ∆H c values of isolated B-subunits were similar to those of the higher-temperature transition peak (B-subunit) of cholera toxin [38]. Similar observations have also been made in the case of several multidomain proteins, which are stabilized by intradomain interactions and not by interdomain interactions [39].…”

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

“…Reduction of the CTA1/CTA2 disulfide bond permits the chaperone-assisted dissociation of CTA1 from CTA2/CTB 5 (7,8). CTA1 is held in a stable conformation by its interactions with the holotoxin (9,10), but the isolated CTA1 polypeptide is an unstable protein that will spontaneously unfold at 37°C upon its separation from CTA2/CTB 5 (11). The disordered CTA1 polypeptide is subsequently recognized as a misfolded protein by the host quality control system of ER-associated degradation (ERAD) (12)(13)(14).…”