Disclose ATP-synthase as a protein target of the antimicrobial peptide RiLK1 in Escherichia coli: An alternative receptor-mediated bactericidal mechanism

Abstract

In this study, we investigated the mechanism of action of the 10-aminoacid RiLK1 peptide against Escherichia coli (strain ATCC 25922), both in vitro and in contaminated meat matrices. Therefore, a mass spectrometry-based functional proteomics platform was employed to identify specific molecular targets of RiLK1 in a membrane protein-enriched E. coli lysate and to obtain information on their interaction mechanism. This target deconvolution approach combines MS-limited proteolysis techniques, like Drug Affinity Responsive Target Stability (DARTS) and targeted-limited Proteolysis coupled with Mass Spectrometry (t-LiP-MS). The b and δ subunits of the multimeric enzymatic complex ATP synthase, the smallest known biological nanomotor found in all cells, were identified as the relevant RiLK1 membrane protein targets. Extensive molecular docking and biochemical analyses validated and improved the suggested interaction profile. These unique findings could rationally explain the relevant RiLK1 bactericidal effects against E. coli strains, suggesting its potential application in food safety and preservation.

Keywords: Antimicrobial peptides; E. coli ATP synthase; Escherichia coli; Food preservation; Functional proteomics; Molecular docking.