title: Two-dimensional structures of the Shiga toxin B-subunit and of a chimera bound to the glycolipid receptor Gb3.
abstract: The B-subunit of Shiga toxin has been demonstrated as a powerful vector for carrying attached peptides into cells for intracellular transport studies and for medical research. We have investigated the structure of the B-subunit and of a chimera bearing a peptide extension, bound to the membranous lipidic receptor, the globotriaosylceramide (Gb3). Two-dimensional crystals of both B-subunits have been obtained by the lipid layer method and projection maps have been calculated at 8.5A resolution from ice-embedded samples. The B-subunits as the chimera are organized in a pentameric form similar to the X-ray structure of the B-subunit not bound to Gb3. A difference map of both proteins has been calculated in which no density could be attributed to the peptide extension. Cross-correlations with projections of the B-subunit X-ray structure revealed that pentamers in the 2D crystals were oriented with their binding sites pointing to the lipid layer. Thus, it is likely that the peptide extension was disordered and confined to the surface of the pentamer opposite to the Gb3 binding sites. This location confirms the hypothesis that addition of peptide extension to the C-terminus conserves the ability of the modified B-subunit to bind the membranous receptor Gb3.
authors.affiliation: Institut Curie, UMR CNRS 168, 11 rue P.M. Curie, F-75231 Paris, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Hagnerelle
authors.forename: Célia
authors.initials: C
authors.lastname: Plisson
authors.forename: Olivier
authors.initials: O
authors.lastname: Lambert
authors.forename: Sergio
authors.initials: S
authors.lastname: Marco
authors.forename: Jean Louis
authors.initials: JL
authors.lastname: Rigaud
authors.forename: Ludger
authors.initials: L
authors.lastname: Johannes
authors.forename: Daniel
authors.initials: D
authors.lastname: Lévy
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D008051:Lipid Bilayers / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D022621:Shiga Toxin / Q000737:chemistry*; D014281:Trihexosylceramides / Q000737:chemistry*
chemical_list: D008051:Lipid Bilayers; D010455:Peptides; D014281:Trihexosylceramides; C018549:globotriaosylceramide; D022621:Shiga Toxin