title: 1H,13C,15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding.
abstract: The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB5toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the1H,13C,15N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on13C',13Cα, 13Cβ, 1HNand15N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM1-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens.
authors.affiliation: Department of Chemistry, University of Oslo, Blindern, P.O. Box 1033, 0315, Oslo, Norway.|Department of Biosciences, University of Oslo, Blindern, P.O. Box 1066, 0316, Oslo, Norway.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hatlem
authors.affiliation: Department of Chemistry, University of Oslo, Blindern, P.O. Box 1033, 0315, Oslo, Norway.|School of Pharmacy, University of Oslo, Blindern, P.O. Box 1068, 0316, Oslo, Norway.
authors.forename: Julie E
authors.initials: JE
authors.lastname: Heggelund
authors.affiliation: Department of Chemistry, University of Oslo, Blindern, P.O. Box 1033, 0315, Oslo, Norway.|Department of Molecular and Cell Biology, University of Leicester, University Road, Leicester, LE1 7RH, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Burschowsky
authors.affiliation: Department of Chemistry, University of Oslo, Blindern, P.O. Box 1033, 0315, Oslo, Norway. ute.krengel@kjemi.uio.no.
authors.forename: Ute
authors.initials: U
authors.lastname: Krengel
authors.affiliation: Department of Biosciences, University of Oslo, Blindern, P.O. Box 1066, 0316, Oslo, Norway. p.e.kristiansen@ibv.uio.no.
authors.forename: Per E
authors.initials: PE
authors.lastname: Kristiansen
mesh_terms: D000595:Amino Acid Sequence; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D004768:Enterotoxins; D009844:Oligosaccharides; C000624194:neolactotetraose
keywords: ETEC; LTB; Molecular recognition; Neolactotetraose; Protein-carbohydrate interactions