title: Structural characteristics of the plasmid-encoded toxin from enteroaggregative Escherichia coli.
abstract: 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 degrees C, with significant thermal unfolding only occurring at temperatures >or=50 degrees C. Fluorescence quenching experiments detected the partial solvent exposure of Pet aromatic amino acid residues at 37 degrees 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.
authors.affiliation: Department of Molecular Biology and Microbiology, Burnett School of Biomedical Science, College of Medicine, University of Central Florida, 12722 Research Parkway, Orlando, Florida 32826, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Scaglione
authors.forename: Kathleen N
authors.initials: KN
authors.lastname: Nemec
authors.forename: Kaitlin E
authors.initials: KE
authors.lastname: Burlingame
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Grabon
authors.forename: Jazmin
authors.initials: J
authors.lastname: Huerta
authors.forename: Fernando
authors.initials: F
authors.lastname: Navarro-Garcia
authors.forename: Suren A
authors.initials: SA
authors.lastname: Tatulian
authors.forename: Ken
authors.initials: K
authors.lastname: Teter
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002942:Circular Dichroism / Q000379:methods; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C530786:Pet protein, E coli; D012697:Serine Endopeptidases