Folding intermediates of SNARE complex assembly

SNARE (soluble NSF attachment protein receptor) proteins assemble into a st able complex essential for vesicle-membrane fusion. To further understand S NARE function we have used solution nuclear magnetic resonance (NMR) spectr oscopy to characterize three assembly states of a yeast SNARE complex: firs t, the 'closed' conformation of Ssol; second, the binary complex of Sso1 an d Sec9; and third, the ternary complex of Ssol, Sec9 and Snc1. Sec9 and Snc 1 are unstructured in isolation. Ssol likely consists of a four helix bundl e 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. Confor mational switching of the H-core domain, via HAHBHC, may provide a key regu latory mechanism in SNARE assembly. Formation of binary and ternary complex es induces additional alpha-helical structure in previously unstructured re gions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into clos e proximity and possibly leading to membrane fusion.