Cyclic peptides identified by phage display are competitive inhibitors of the tRNA-dependent amidotransferase of Helicobacter pylori

• The tRNA-dependent amidotransferase GatCAB is essential for Helicobacter pylori.
• GatCAB participates in the biosynthesis of Gln-tRNAGln and/or Asn-tRNAAsn in H. pylori.
• Several cyclic peptides identified by phage display inhibit H. pylori GatCAB.
• P10 (CMPVWKPDC) is a competitive inhibitor of GatCAB with respect to Glu-tRNAGln.
• Molecular docking suggests that Trp5 and Lys6 of P10 are important for GatCAB binding.

In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNAGln and Asp-tRNAAsn into Gln-tRNAGln and Asn-tRNAAsn, 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-tRNAGln, 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-tRNAGln, with Ki values of 126 and 392 μM, 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-tRNAGln 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-tRNAGln and/or Asn-tRNAAsn formation.

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