Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin

Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca2+ could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca2+-dependent calmodulin binding (K-d = 500 nM) to glutathione S-transferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP(77-94), immediately C-terminal to the tetanus toxin cleavage site (Q(76-F77)) In isolated synaptic vesicles, Ca2+/calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin, Assembly of a S-35-SNAP-25, syntaxin I GST-VAMP(1-96) complex was inhibited by Ca2+/calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP77-94 and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in alpha-helical content in a hydrophobic: environment. These data provide insight into the mechanisms by which Ca2+ may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.