Stabilization of the Tertiary Structure of the Cholera Toxin A1 Subunit Inhibits Toxin Dislocation and Cellular Intoxication

Abstract

Summary Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit - specifically, the loss of CTA1 tertiary structure at 37°C - triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel anti-toxin therapeutic agents.

How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum

“…Consistent with this, in vivo stabilisation of the structure of CTxA1 with glycerol reduces cholera toxicity by inhibiting dislocation (Massey et al 2009). Furthermore, dissection of the process of unfolding reveals subdomains of A1 that are temperaturesensitive and those that serve to stabilise A1 structure, studies that may lead to a full mechanistic description of the unfolding process (Banerjee et al 2010).…”

Molecular Dynamics Simulation of Cholera Toxin A-1 Polypeptide

“…The secondary structure of cholera toxin is of great interest because of its unusual entry mechanism of translocation inside the cell cytosol [44][45][46]. It utilizes the retrotranslocation mechanism and deceives the proteolytic enzymes of endoplasmic reticulum and also uses the process of unfolding and refolding mechanism [15,17,20].…”

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

“…Filipin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); CT was purchased from List Biologicals (Campbell, CA); and isoproterenol was purchased from Sigma-Aldrich. CTA1 with a C-terminal His 6 tag was purified as described previously (13) and used for all in vitro studies.…”

“…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). This results in the ER-to-cytosol translocation of CTA1 through one or more protein-conducting channels in the ER membrane (15)(16)(17)(18).…”