Interaction of Synaptotagmin with Lipid Bilayers, Analyzed by Single-Molecule Force Spectroscopy

AbstractSynaptotagmin I is the major Ca2+ sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca2+, the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca2+-dependent and Ca2+-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca2+. The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca2+-independent force depends only on C2A. When the lysine residues K189–192 and K326, 327 were mutated to alanine, the strong Ca2+-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca2+, and also that positively charged regions of both C2A and C2B are essential for the strong Ca2+-dependent binding of synaptotagmin to the bilayer