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

Abstract

Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin. Cholera toxin (CT)2 is an AB 5 protein toxin that consists of a catalytic A moiety and a cell-binding B moiety (1, 2). The B subunit is pentameric ring-like structure that adheres to GM1 gangliosides on the plasma membrane of a target cell. The A subunit is initially synthesized as a 26 kDa protein that undergoes proteolytic nicking to generate a disulfide-linked A1/A2 heterodimer. The 21 kDa CTA1 polypeptide is an ADP-ribosyltransferase that modifies and activates Gs␣ in the host cell cytosol. CTA1 can be divided into three subdomains: the A1 1 subdomain contains the catalytic core of the toxin; the A1 2 subdomain is a short extended linker that connects the A1 1 and A1 3 subdomains; and the A1 3 subdomain is a globular structure with many hydrophobic residues as well as a cysteine residue involved with the single disulfide bridge between CTA1 and CTA2 (3). The 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the central pore of the B pentamer and thereby anchors CTA1 to CTB 5 . A ribbon diagram of the CT holotoxin which highlights the subdomain structure of CTA1 is provided in supplemental Fig. S1.To reach its cytosolic Gs␣ target, CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular traffic (4). A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4, 5). Conditions in the ER lead to reductive cleavage of the CTA1/CTA2 disulfide bond and chaperone-assisted dissociation of CTA1 from CTA2/CTB 5 (6 -9). Unfolding of the free A1 subunit then activates the quality control system of ER-associated degradation (ERAD), thereby promoting CTA1 translocation to the cytosol (10, 11). Most exported ERAD substrates are degraded by the ubiquitin-proteasome system, but it was hypothesized that CTA1 and the A chains of other ER-translocating toxins avoid t...

Molecular Dynamics Simulation of Cholera Toxin A-1 Polypeptide

“…From these results we concluded that CTA1 has partially disordered secondary structure and can be placed in the intrinsically disordered (ID) protein class. The behavior of cholera toxin A1 polypeptide of regular units of helices and β-sheets interrupted by coils were in close agreement to the work of Ampapathi et al [16] who observed the secondary structure through circular dichroism spectroscopy and 2D nuclear magnetic resonance spectroscopy and that of others [13][14][15][16].…”

“…The first 132 amino acids formed a compact globular unit composed of a mixture of α-helices and β-strands [10,12,13]. The catalysis is thought to occur in well define fissure on the free surface of A-subunit [10,12,14].…”

“…This enzyme breaks the disulfide bond between amino acid residues 187 and 199, splitting the A-subunit into A1 and A2 chains. The A1 chain of CT unfolds inside the endoplasmic reticulum and then enters the cytosol by hijacking the cellular machinery that enables misfolded proteins to cross the membrane for degradation by the proteasome, a process termed retro-translocation [14]. In the cytosol the partially unfolded CTA1 has limited in vivo activity and is prone to ubiquitin independent proteasomal degradation, so CTA1 binds to ARF6 (cytosolic protein) forming cholera toxin A-1 subunit Adenosine diphosphoribosyl factor 6 Guanosine triphosphate (CTA1-ARF6-GTP) complex for refolding and stability [11,[17][18][19].…”

“…In the endoplasmic reticulum the A-subunit breaks into A1 and A2 chains by the action of protein disulfide isomerase (PDI) [14]. This enzyme breaks the disulfide bond between amino acid residues 187 and 199, splitting the A-subunit into A1 and A2 chains.…”

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

“…The disulfide bond linking CTA1 to CTA2 must then be reduced (4), which only occurs after the CT holotoxin moves from the cell surface to the endoplasmic reticulum (ER) through a series of vesicle-mediated trafficking steps collectively termed retrograde transport (5,6). 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).…”