Somiya, M.; Funk, S.; Zambrano, D.; Yanase, T.; Hamaoka, N.; Kang, A.; Sankaran, B.; Bera, A. K.; King, N. P.

The fusion of two distinct biological membranes is an evolutionarily conserved process essential to cellular organization and physiology. Membrane fusion is driven by the refolding of fusogenic proteins into low-energy postfusion states that overcome the energetic barrier to bilayer merger. Here we report a computational method for the design of synthetic fusogens inspired by the architecture of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Using machine learning-guided protein design to extensively remodel backbone geometry and sequence, we generated heterodimeric SNARE-like assemblies that efficiently catalyze cell-cell membrane fusion. These minimal two-component fusogens exhibit substantially higher fusion activity than native multisubunit SNARE complexes. Structural and functional analyses identify the key determinants required for fusogenic activity and reveal a modularity that enables control of fusion through chemically induced heterodimerization. In addition to cell-cell fusion, the synthetic fusogens drive fusion between endoplasmic reticulum and mitochondrial membranes from human cells, demonstrating their potential as tools for programmable manipulation of intracellular membranes. Together, these results establish a general framework for the rational design of synthetic fusogens and expand the toolkit for engineering membrane dynamics in living systems.