File(s) not publicly available
A pH-dependent conformational change in the B-subunit pentamer of Escherichia coli heat-labile enterotoxin: Structural basis and possible functional role for a conserved feature of the AB5 toxin family
journal contribution
posted on 2023-06-10, 04:07 authored by Lloyd W Ruddock, Helen WebbHelen Webb, Stephen P Ruston, Caroline Cheesman, Robert B Freedman, Timothy R HirstThe non-covalently associated B-subunit moieties of AB5 toxins, such as cholera toxin and related diarrheagenic enterotoxins, exhibit exceptional pH stability and remain pentameric at pH values as low as 2.0. Here, we investigate the structural basis of a pH-dependent conformational change which occurs within the B5 structure of Escherichia coli heat-labile enterotoxin (EtxB) at around pH 5.0. The use of far-UV CD and fluorescence spectroscopy showed that EtxB pentamers undergo a fully reversible pH-dependent conformational change with a pKa of 4.9 ± 0.1 (R2 = 0.999) or 5.13 ± 0.01 (R2 = 0.999), respectively. This renders the pentamer susceptible to SDS-mediated disassembly and decreases its thermal stability by 18 °C. A comparison of the pH-dependence of the structural change in EtxB5, with that of a mutant containing a Ser substitution at His 57, revealed that the pKa of the conformational change was shifted from ca. 5.1 to 4.4. This finding suggests that protonation of the imidazole side chain of His 57 might facilitate disruption of a spatially adjacent salt bridge, located between Glu 51 and Lys 91 in each B-subunit, thus triggering the conformational change in the pentameric structure. The pH-dependent conformational change was found to be inhibited when B-subunits bound to monosialoganglioside, GM1; and to have no effect on the stability of interaction between A- and B-subunits within the AB5 complex. This suggests that the conformational change is unlikely to have a direct involvement in toxicity. Conservation of the pH-dependent conformational change in the AB5 toxin family, combined with the potential exposure of the hydrophobic core of ß-barrel in the monomeric units, leads to the proposal that the conformational change may be the common feature that ensures the secretion of these proteins from the Vibrionaceae.
History
Publication status
- Published
Journal
BiochemistryISSN
0006-2960Publisher
American Chemical Society (ACS)External DOI
Volume
35Page range
16069-16076Event location
United StatesDepartment affiliated with
- Biochemistry Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2022-07-01Usage metrics
Categories
No categories selectedKeywords
Amino Acid Sequence;Animals;Bacterial Toxins;Cholera Toxin;Conserved Sequence;ElectrophoresisPolyacrylamide Gel;Enterotoxins;Escherichia coli;Escherichia coli Proteins;Histidine;Humans;Hydrogen-Ion Concentration;Kinetics;Lysine;Macromolecular Substances;ModelsMolecular;Protein Conformation;Swine;Tryptophan
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC