Background: We previously reported the identification of the aapA1/IsoA1 locus as part of a new family of toxin-antitoxin (TA) systems in the human pathogen Helicobacter pylori. AapA1 belongs to type I TA bacterial toxins, and both its mechanism of action towards the membrane and toxicity features are still unclear.
Methods: The biochemical characterization of the AapA1 toxic peptide was carried out using plasmid-borne expression and mutational approaches to follow its toxicity and localization. Biophysical properties of the AapA1 interaction with lipid membranes were studied by solution and solid-state NMR spectroscopy, plasmon waveguide resonance (PWR) and molecular modeling.
Results: We show that despite a low hydrophobic index, this toxin has a nanomolar affinity to the prokaryotic membrane. NMR spectroscopy reveals that the AapA1 toxin is structurally organized into three distinct domains: a positively charged disordered N-terminal domain (D), a single α-helix (H), and a basic C-terminal domain (R). The R domain interacts and destabilizes the membrane, while the H domain adopts a transmembrane conformation. These results were confirmed by alanine scanning of the minimal sequence required for toxicity.
Conclusion: Our results have shown that specific amino acid residues along the H domain, as well as the R domain, are essential for the toxicity of the AapA1 toxin.
General significance: Untangling and understanding the mechanism of action of small membrane-targeting toxins are difficult, but nevertheless contributes to a promising search and development of new antimicrobial drugs.
Keywords: Bacterial toxin; Helicobacter pylori; Small membrane protein; Toxin-antitoxin; Transmembrane domain.
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