Modulation of cholera toxin structure and function by host proteins

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process

Adp-Ribosylation Factor 6 Acts As An Allosteric Activator For The Folded But Not Disordered Cholera Toxin A1 Polypeptide

The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP-ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope-edited Fourier transform infrared spectroscopy monitored ARF6-induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF-mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells

Refolding of disordered PDI.

<p>PDI was placed at 10°C in sodium borate buffer (pH 7.0) containing ¹³C-labeled CTA1 and 1 mM GSH. The temperature was then raised to 37°C for 60 min. (<b>A</b>) The FTIR spectra of PDI+CTA1 were recorded at 10°C (solid line) and at 37°C (dotted line). (<b>B</b>) Curve fitting (left panel) and second derivatives (right panel) for the FTIR spectrum of PDI at 37°C are shown. For curve fitting, the dotted line represents the sum of all deconvoluted components (solid lines) from the measured spectrum (dashed line).</p

The chaperone activity of PDI is required for CT intoxication.

<p>(<b>A</b>) Untreated and ribostamycin-treated CHO cells were challenged with the stated concentrations of CT for 2 hr before cAMP levels were quantified. The averages ± ranges of 2 independent experiments with triplicate samples are shown. (<b>B</b>) CHO cells were transfected with a plasmid encoding a CTA1 construct that is co-translationally inserted into the ER. Dislocation of this CTA1 construct back to the cytosol of untreated or ribostamycin-treated cells was detected by the rise in intracellular cAMP at 4 hr post-transfection. Cells transfected with an empty plasmid (Mock) were used to establish the resting levels of cAMP. Data are presented as the averages ± standard deviations of three replicate samples per condition. One of three representative experiments is shown. (<b>C</b>) CHO cells were pulse-labeled at 4°C with 1 µg/mL of CT. Untreated or drug-treated cells were then chased in toxin-free medium for 2 hr at 37°C. Membrane fractions from digitonin-permeabilized cells were resolved by non-reducing SDS-PAGE and probed by Western blot with an anti-CTA antibody. (<b>D</b>) Untreated or ribostamycin-treated CHO cells were pulse-labeled at 4°C with 1 µg/mL of CT and then chased in toxin-free medium for 2 hr at 37°C. Cytosolic fractions from digitonin-permeabilized cells were then perfused over an SPR sensor coated with an anti-CTA1 monoclonal antibody. Known quantities of CTA were perfused over the sensor as positive controls, and the cytosolic fraction from unintoxicated cells was perfused over the sensor as a negative control. At the end of each perfusion, bound ligand was stripped from the sensor slide.</p

Loss of PDI structure in the presence of CTA1.

<p>Far-UV CD spectra of PDI (green), CTA1 (blue), and both proteins in the same sample at a 1∶1 molar ratio (red) are shown. The black dotted line was obtained by spectral subtraction of the individual CTA1 and PDI spectra from the spectrum of both proteins together. All measurements were taken at 10°C in pH 7.0 buffer containing 1 mM GSH.</p

PDI unfolding but not oxidoreductase activity is required for disassembly of the CT holotoxin.

<p>SPR was used to monitor the real-time PDI-mediated disassembly of CT. A baseline measurement corresponding to the mass of the sensor-bound CT holotoxin established the 0 MicroRIU signal. The time course was then initiated with perfusion of PDI (<b>A</b>), EDC-treated PDI (<b>B</b>), or bacitracin-treated PDI (<b>C</b>) over the CT-coated sensor. The perfusion buffer contained either 30 mM GSH (left panels) or 1 mM GSH (right panels); non-reducing SDS-PAGE with Coomassie staining found the CTA1/CTA2 disulfide bond was reduced at 30 mM GSH but not 1 mM GSH (<i>inset</i>, right panel of <b>A</b>). PDI was removed from the perfusion buffer at time intervals denoted by asterisks and was replaced with sequential additions of anti-PDI, anti-CTA1, and anti-CTB antibodies as indicated by the arrowheads. One of two representative experiments is shown for each condition.</p

Structure of PDI in the absence or presence of CTA1.

<p>Curve fitting (left panels) and second derivatives (right panels) for the FTIR spectrum of PDI recorded in the absence (<b>A</b>, <b>C</b>, <b>E</b>, <b>G</b>) or presence (<b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>) of ¹³C-labeled CTA1 are shown. For curve fitting, the dotted line represents the sum of all deconvoluted components (solid lines) from the measured spectrum (dashed line). Unless otherwise noted, all experiments were performed with sodium borate buffer (pH 7.0) containing 1 mM GSH. (<b>A</b>, <b>B</b>) PDI structure at 10°C. (<b>C</b>, <b>D</b>) PDI structure at 10°C in the absence of reductant. (<b>E</b>, <b>F</b>) PDI structure at 37°C. (<b>G</b>, <b>H</b>) PDI structure at 37°C in pH 6.5 buffer.</p

The chaperone activity of PDI is required for disassembly of the CT holotoxin.

<p>(<b>A</b>, <b>B</b>) A baseline SPR measurement corresponding to the mass of the sensor-bound CT holotoxin established the 0 MicroRIU signal. The time course was then initiated with perfusion of S-nitrosylated PDI (<b>A</b>) or ribostamycin-treated PDI (<b>B</b>) over the CT-coated sensor in buffer containing 30 mM GSH. In (<b>B</b>), PDI was removed from the perfusion buffer after 600 sec and replaced with sequential additions of anti-PDI, anti-CTA1, and anti-CTB antibodies as indicated by the arrowheads. One of two representative experiments is shown for each condition. (<b>C</b>, <b>D</b>) Curve fitting (left panels) and second derivatives (right panels) for the FTIR spectrum of ribostamycin-treated PDI recorded in the absence (<b>C</b>) or presence (<b>D</b>) of ¹³C-labeled CTA1 are shown. For curve fitting, the dotted line represents the sum of all deconvoluted components (solid lines) from the measured spectrum (dashed line).</p