Structural Characteristics Of The Plasmid-Encoded Toxin From Enteroaggregative Escherichia Coli

Intoxication by the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli requires toxin translocation from the endoplasmic reticulum (ER) to the cytosol. This event involves the quality control system of ER-associated degradation (ERAD), but the molecular details of the process are poorly characterized. For many structurally distinct AB-type toxins, ERAD-mediated translocation is triggered by the spontaneous unfolding of a thermally unstable A chain. Here we show that Pet, a non-AB toxin, engages ERAD by a different mechanism that does not involve thermal unfolding. Circular dichroism and fluorescence spectroscopy measurements demonstrated that Pet maintains most of its secondary and tertiary structural features at 37°C, with significant thermal unfolding only occurring at temperatures ≥50°C. Fluorescence quenching experiments detected the partial solvent exposure of Pet aromatic amino acid residues at 37°C, and a cell-based assay suggested that these changes could activate an ERAD-related event known as the unfolded protein response. We also found that HEp-2 cells were resistant to Pet intoxication when incubated with glycerol, a protein stabilizer. Altogether, our data are consistent with a model in which ERAD activity is triggered by a subtle structural destabilization of Pet and the exposure of Pet hydrophobic residues at physiological temperature. This was further supported by computer modeling analysis, which identified a surface-exposed hydrophobic loop among other accessible nonpolar residues in Pet. From our data it appears that Pet can promote its ERAD-mediated translocation into the cytosol by a distinct mechanism involving partial exposure of hydrophobic residues rather than the substantial unfolding observed for certain AB toxins. © 2008 American Chemical Society

Inelastic Scattering of a Photon by a Quantum Phase Slip

International audienc

Inelastic Scattering of a Photon by a Quantum Phase Slip

International audienc