title: Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin.
abstract: Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca(2+) could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca(2+)-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)-F(77)). In isolated synaptic vesicles, Ca(2+)/calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a (35)S-SNAP-25, syntaxin 1 GST-VAMP(1-96) complex was inhibited by Ca(2+)/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 VAMP(77-94) and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in alphahelical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca(2+) may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale Unité, Université de la Méditerrannée, Marseille, France.
authors.forename: S
authors.initials: S
authors.lastname: Quetglas
authors.forename: C
authors.initials: C
authors.lastname: Leveque
authors.forename: R
authors.initials: R
authors.lastname: Miquelis
authors.forename: K
authors.initials: K
authors.lastname: Sato
authors.forename: M
authors.initials: M
authors.lastname: Seagar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D005089:Exocytosis; D008081:Liposomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017433:Protein Structure, Secondary; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D013744:Tetanus Toxin / Q000494:pharmacology; D033921:Vesicular Transport Proteins
chemical_list: D000954:Antigens, Surface; D002147:Calmodulin; D008081:Liposomes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010718:Phosphatidylserines; D010743:Phospholipids; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; D002118:Calcium