Folding intermediates of SNARE complex assembly

SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Ssol; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Ssol, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Ssol likely consists of a four helix bundle formed by part of the C-terminal H-core domain and the N-terminal HAHBHC domain, and this bundle is flanked on both sides by large flexible regions. Ssol switches to an 'open' state when its H-core domain binds Sec9. Conformational switching of the H-core domain, via HAHBHC, may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.