Engineered Coiled-Coils Convert Cholera Toxin B‑Pentamers into Programmable Membrane Fusogens

Membrane fusion is central to biological function and bioengineering, yet few design rules exist that enable proteins to be programmed to drive fusion at defined membrane interfaces. Here, we show 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 fusion 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; strikingly, fusogenic efficiency was governed primarily by the length of the CTA2 linker rather than coiled-coil orientation. These findings establish a generalizable strategy for engineering lectin-based fusogens with tunable activity, defining linker geometry as a key design parameter and advancing the development of programmable membrane fusion platforms for drug delivery and synthetic cell systems.