Membrane-gated SNARE zippering focuses energy for fusion

Soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) drive stagewise membrane fusion by zippering into membrane-bridging four-helix bundles. Yet the conformations underlying successive fusion stages and the coupling of folding energy to bilayer remodeling remain unclear. Using optical tweezers, we measured the intermediates, energetics, kinetics, and force dependence of individual synaptic SNARE complexes assembled in cis on single membranes and in trans between apposed membranes. Membrane-anchored cis-SNAREs assembled through N-terminal and cooperative C-terminal/linker-domain transitions, whereas their transmembrane domains showed little intrinsic dimerization. Syntaxin retained membrane-dependent helical continuity through its linker domain before zippering was complete. PIP₂ strengthened but slowed late zippering. In trans, membrane repulsion arrested single trans-SNARE complexes in a half-zippered state. Gβγ further clamped this intermediate and inhibited late zippering; Gα–GDP, but not Gα–GTPγS, relieved the clamp, revealing a nucleotide-dependent mechanism for GPCR-mediated inhibition of neurotransmitter release. Modeling suggests that cooperative late zippering, syntaxin linker helicity, and concerted action of multiple SNAREs focus folding energy released over a long distance onto short-range membrane apposition. Thus, mechanically gated SNARE zippering is regulated by membrane forces, lipids, and regulatory proteins.