Engineered coiled-coils convert cholera toxin B-pentamers into programmable membrane fusogens

ABSTRACT

Membrane fusion underpins many fundamental cellular processes, yet the design of artificial fusogens that can interact with specific cell surface markers in a precise and predictable manner remains a major challenge. Here we demonstrate that cholera toxin B-subunit (CTB), a naturally occurring glycolipid-binding pentamer, can be re-engineered into a programmable membrane fusogen by assembling two CTB units through rationally designed coiled-coil linkers attached to the CTA2 peptide that threads through the CTB pentamer. Using discrete parallel and antiparallel coiled-coil architectures, we generated CTB dimers with defined orientations and examined their ability to drive fusions of giant unilamellar vesicles containing the CTB ligand ganglioside GM1. Fusion efficiency was evaluated using a fluorescence resonance energy transfer (FRET)-based lipid mixing assay, while flow cytometry, confocal microscopy and quartz crystal microbalance with dissipation monitoring (QCM-D) provided mechanistic insights. Both parallel and antiparallel CTB dimers induced cross-linking and full fusion of vesicles, with the efficiency of fusion governed primarily by the length of the CTA peptide linker rather than coiled-coil orientation. These findings establish a robust strategy to engineer lectin-based fusogens with tunable activity, providing new opportunities for the development of targeted membrane fusion systems for drug delivery and synthetic biology.