An atypical V-shaped garvicin C bacteriocin targets Man-PTS via domain insertion

Bacteriocins are ribosomally synthesized antimicrobial peptides that often rely on specific membrane receptors for activity. Among these receptors, the mannose-specific phosphotransferase system (Man-PTS) is a conserved bacterial target for structurally diverse peptides. Although most Man-PTS-binding bacteriocins share a canonical β-sheet/α-helix fold, members of the GarC-like subgroup exhibit an atypical structure composed entirely of α-helices with an unstructured C-terminal region. Here, we combine site-directed mutagenesis and antimicrobial assays with AlphaFold2 modeling and molecular dynamics simulations to define the structural basis of GarC recognition of Man-PTS. We identify conserved residues critical for receptor interaction and demonstrate that GarC's V-shaped α-helical core inserts between the Vmotif and Core domains of Man-PTS. Functional data support the model: substitutions in GarC or Man-PTS that most strongly alter antimicrobial potency and receptor sensitivity cluster at the predicted contact residues. Molecular dynamics further reveals that GarC binding induces transmembrane, solvent-accessible tunnels consistent with a pore-forming mechanism, which we confirm experimentally using membrane-permeabilization assays. These findings reveal a distinct mode of Man-PTS engagement and provide a framework for rational design of selective, low-toxic antimicrobials.