# The "Disaggregase" Mechanism of Holotoxin Disassembly by Protein Disulfide Isomerase

> **NIH NIH R01** · UNIVERSITY OF CENTRAL FLORIDA · 2020 · $154,672

## Abstract

Cholera toxin (CT) is an AB5 toxin that consists of a catalytic A1 subunit, an A2 linker, and a cell-binding B
pentamer. The separation of CTA1 from CTA2/CTB5 is required for in vivo toxin activity. This occurs after the
holotoxin travels by vesicle carriers from the plasma membrane to the endoplasmic reticulum (ER) of a target
cell. Reduction of the CTA1/CTA2 disulfide bond occurs at the resident redox state of the ER, but the reduced
toxin remains intact. CTA1 must be actively displaced from its non-covalent assembly in the reduced holotoxin
by protein disulfide isomerase (PDI), an ER-localized protein with linked but distinct functions as a chaperone
and oxidoreductase. The free A1 subunit then moves from the ER to the cytosol where it initiates the cellular
events leading to a profuse watery diarrhea that causes 1-4 million illnesses and 100,000 deaths per year.
The goal of this project is to define the molecular details of an essential but poorly understood event in cholera
intoxication: PDI-mediated holotoxin disassembly. Our recent biophysical analysis has provided the foundation
to understand this process. We have shown by isotope-edited Fourier transform infrared (FTIR) spectroscopy
that PDI unfolds upon contact with CTA1. A real-time holotoxin disassembly assay demonstrated the
displacement of reduced CTA1 from CTA2/CTB5 does not occur when PDI is locked in a folded conformation
or when its chaperone function is disrupted by drug treatment. In contrast, the oxidoreductase activity of PDI is
not required for CT disassembly. The partial unfolding of PDI provides a molecular basis for CT disassembly:
the expanded hydrodynamic size of unfolded PDI would push against two components of the CT holotoxin,
thus acting as a wedge to dislodge reduced CTA1 from the rest of the toxin. This phenomenon could also
apply to PDI interactions with other AB toxins, and it provides a basis for the established but structurally
uncharacterized neuroprotective chaperone activity of PDI: by unfolding in the presence of an amyloid-forming
substrate, PDI would act as a “disaggregase” to displace individual proteins from the neurotoxic aggregate.
PDI has an abb'xa' organization that consists of two thioredoxin-like catalytic domains (a & a') separated by
two non-catalytic domains (b & b') and an x linker. Based on preliminary and published data, we predict CTA1
binding to the b domain of PDI transmits a signal through the b'x domains for unfolding of the a' domain. We
further predict that PDI binds to a region of CTA1 that positions its a' domain near the interface of CTA1 and
CTA2. Unfolding of the a' domain would then create a wedge between CTA1 and CTA2, leading to the release
of CTA1 from its reduced holotoxin. Interrogation of this model will provide detailed mechanistic insight into the
unique and previously unrecognized “disaggregase” activity of PDI that is responsible for CT disassembly, with
potentially broad relevance to toxin biology, neurobiology, and...

## Key facts

- **NIH application ID:** 10214345
- **Project number:** 3R01AI137056-03S1
- **Recipient organization:** UNIVERSITY OF CENTRAL FLORIDA
- **Principal Investigator:** KENNETH R TETER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $154,672
- **Award type:** 3
- **Project period:** 2018-02-03 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10214345

## Citation

> US National Institutes of Health, RePORTER application 10214345, The "Disaggregase" Mechanism of Holotoxin Disassembly by Protein Disulfide Isomerase (3R01AI137056-03S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10214345. Licensed CC0.

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