title: Analysis of structure-function relationships in the colibactin-maturating enzyme ClbP.
abstract: pks genomic island of Escherichia coli is involved in the synthesis of the non-ribosomal peptide-type genotoxin colibactin, which has been suggesting as affecting the host immune response and having an impact on cancer development. The pks-encoded enzyme ClbP is an atypical peptidase that contributes to the synthesis of colibactin. In this work, we identified key features of ClbP. Bacterial fractionation and Western-blot analysis revealed the docking of ClbP to the bacterial inner membrane via a C-terminal domain harboring three predicted transmembrane helices. Whereas only one helix was necessary for the location in the inner membrane, the complete sequence of the C-terminal domain was necessary for ClbP bioactivity. In addition, the N-terminal sequence of ClbP allowed the SRP/Sec/YidC- and MreB-dependent translocation of the enzymatic domain in the periplasmic compartment, a feature also essential for ClbP bioactivity. Finally, the comparison of ClbP structure with that of the paralogs FmtA-like and AmpC revealed at an extremity of the catalytic groove a negative electrostatic potential surface characteristic of ClbP. Site-directed mutagenesis experiments identified in this zone two aspartic residues that were important for ClbP bioactivity. Overall, these results suggest a model for precolibactin activation by ClbP and pave a way for the design of inhibitors targeting colibactin production.
authors.affiliation: M2iSH, INSERM U1071, INRA USC2018, Clermont Université, Université d'Auvergne, 63000 Clermont-Ferrand, France.
authors.forename: Antony
authors.initials: A
authors.lastname: Cougnoux
authors.forename: Lucie
authors.initials: L
authors.lastname: Gibold
authors.forename: Frederic
authors.initials: F
authors.lastname: Robin
authors.forename: Damien
authors.initials: D
authors.lastname: Dubois
authors.forename: Nathalie
authors.initials: N
authors.lastname: Pradel
authors.forename: Arlette
authors.initials: A
authors.lastname: Darfeuille-Michaud
authors.forename: Guillaume
authors.initials: G
authors.lastname: Dalmasso
authors.forename: Julien
authors.initials: J
authors.lastname: Delmas
authors.forename: Richard
authors.initials: R
authors.lastname: Bonnet
mesh_terms: D015153:Blotting, Western; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D061065:Polyketides / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010455:Peptides; D061065:Polyketides; C569566:colibactin; C577770:ClbP protein, E coli; D010447:Peptide Hydrolases