Order–Disorder–Order Transitions Mediate the Activation of Cholera Toxin

Abstract

Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Delta delta(coil)) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD(+), which revealed that the formation of the complex does not significantly enhance NAD(+) binding. Partial unfolding of CTA1 is further illustrated by using 4,4'-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G(s)alpha, triggering the downstream events associated with the pathophysiology of cholera.

Molecular Dynamics Simulation of Cholera Toxin A-1 Polypeptide

“…The CT and related AB 5 toxins of other bacteria move from the plasma membrane through trans-Golgi and endoplasmic reticulum (ER) to the cytosol of host cells [6,15,16]. In the endoplasmic reticulum the A-subunit breaks into A1 and A2 chains by the action of protein disulfide isomerase (PDI) [14].…”

“…These include the blocking of the assembly of holotoxin, preventing receptor-recognition process and directly targeting the enzymatic active site in the folding, unfolding states [10][11][12]19]. Cholera toxin B-subunit is being used as a drug carrier and is also one of the medically important bacterial toxins and requires extra attention to explore its structural properties [16,24,25]. The aim and objective of this study is to gain an understanding into the structure of the CTA1 subunit, by performing MD simulations.…”

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

“…To reach its G protein target, CTA1 undergoes what has been termed an order-disorder-order transition (55). The ordered CTA1 polypeptide travels from the cell surface to the ER as part of an intact holotoxin (6).…”

“…S1, CTA1 1-168 did not interact with either plasma membrane or lipid raft LUVs. CTA1 1-168 maintains the same stability and the same general structure as full-length CTA1 (12,55), so the loss of binding affinity could not be attributed to structural alterations in CTA1 . Thus, the hydrophobic A1 3 subdomain of CTA1 appeared to mediate toxinlipid interactions directly.…”

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

“…Thermally-unfolded CTxA can be degraded in a ubiquitin-independent fashion by the 20S core proteasome (Pande et al 2007), so CTxA1 survival in the cytosol was thought to require rapid spontaneous folding (Rodighiero et al 2002) allowing proteasomal avoidance. Structural studies, though, suggest that a likely route of toxin activation is via substrate-mediated folding in the cytosol with ARF6 stimulating the refolding of the C-terminus of CTx (Ampapathi et al 2008). In addition, the requirement for Hsp90 for CTx toxicity ) may also reflect cytosolic post-dislocation events where Hsp90 is required for maintenance of CTxA1 in the cytosol, protecting the toxin from proteasomal degradation.…”