title: Cyclic peptides identified by phage display are competitive inhibitors of the tRNA-dependent amidotransferase of Helicobacter pylori.
abstract: In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNA(Gln) and Asp-tRNA(Asn) into Gln-tRNA(Gln) and Asn-tRNA(Asn), respectively. In this study, we used a phage library to identify peptide inhibitors of GatCAB. A library displaying loop-constrained heptapeptides was used to screen for phages binding to the purified GatCAB. To optimize the probability of obtaining competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA(Gln), we used that purified substrate in the biopanning process of the phage-display technique to elute phages bound to GatCAB at the third round of the biopanning process. Among the eluted phages, we identified several that encode cyclic peptides rich in Trp and Pro that inhibit H. pylori GatCAB in vitro. Peptides P10 and P9 were shown to be competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA(Gln), with Ki values of 126 and 392μM, respectively. The docking models revealed that the Trp residues of these peptides form π-π stacking interactions with Tyr81 of the synthetase active site, as does the 3'-terminal A76 of tRNA, supporting their competitive behavior with respect to Glu-tRNA(Gln) in the transamidation reaction. These peptides can be used as scaffolds in the search for novel antibiotics against the pathogenic bacteria that require GatCAB for Gln-tRNA(Gln) and/or Asn-tRNA(Asn) formation.
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec G1V 0A6, Canada; Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec G1V 0A6, Canada; The Quebec Network for Research on Protein Function, Structure, and Engineering (PROTEO), Québec G1V 0A6, Canada. Electronic address: van-hau.pham.1@ulaval.ca.
authors.forename: Van Hau
authors.initials: VH
authors.lastname: Pham
authors.affiliation: Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec G1V 0A6, Canada.
authors.forename: Halim
authors.initials: H
authors.lastname: Maaroufi
authors.affiliation: Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec G1V 0A6, Canada; Département de Biologie Médicale, Faculté de Médicine, Université Laval, Québec G1V 0A6, Canada.
authors.forename: Roger C
authors.initials: RC
authors.lastname: Levesque
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec G1V 0A6, Canada; Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec G1V 0A6, Canada; The Quebec Network for Research on Protein Function, Structure, and Engineering (PROTEO), Québec G1V 0A6, Canada. Electronic address: Jacques.Lapointe@bcm.ulaval.ca.
authors.forename: Jacques
authors.initials: J
authors.lastname: Lapointe
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D020134:Catalytic Domain; D016480:Helicobacter pylori / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D019884:Nitrogenous Group Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D010456:Peptides, Cyclic / Q000737:chemistry*; D011485:Protein Binding
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010456:Peptides, Cyclic; D019884:Nitrogenous Group Transferases; C047694:glutamyl-tRNA(Gln) amidotransferase
keywords: Helicobacter pylori; Inhibitor; Molecular docking; Phage display; tRNA-dependent amidotransferase