title: The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.
abstract: Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1-93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.
authors.affiliation: Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 75724 Paris cedex15, France. carolina.varela-chavez@igh.cnrs.fr.
authors.forename: Carolina
authors.initials: C
authors.lastname: Varela Chavez
authors.affiliation: Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 75724 Paris cedex15, France. georges.haustant@pasteur.fr.
authors.forename: Georges Michel
authors.initials: GM
authors.lastname: Haustant
authors.affiliation: Plate-Forme de Biophysique Moléculaires, Institut Pasteur, 75724 Paris cedex15, France. bruno.baron@pasteur.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Baron
authors.affiliation: Plate-Forme de Biophysique Moléculaires, Institut Pasteur, 75724 Paris cedex15, France. patrick.england@pasteur.fr.
authors.forename: Patrick
authors.initials: P
authors.lastname: England
authors.affiliation: Unité de Biochimie des Interactions Macromoléculaires, Institut Pasteur, 75724 Paris cedex15, France. alexandre.chenal@pasteur.fr.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
authors.affiliation: Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 75724 Paris cedex15, France. serge.pauillac@pasteur.fr.
authors.forename: Serge
authors.initials: S
authors.lastname: Pauillac
authors.affiliation: Unité de Bioinformatique Structurale, Institut Pasteur, 75724 Paris cedex15, France. ablondel@pasteur.fr.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Blondel
authors.affiliation: Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 75724 Paris cedex15, France. mpopoff@pasteur.fr.
authors.forename: Michel-Robert
authors.initials: MR
authors.lastname: Popoff
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001427:Bacterial Toxins; D010718:Phosphatidylserines; C072292:lethal toxin LT, Clostridium sordellii; D020559:Monomeric GTP-Binding Proteins
keywords: C. sordellii lethal toxin; Clostridium difficile; Clostridium sordellii; Ras; large clostridial glucosylating toxin; phosphatidylserine