Structural Characterization of Full-Length Synaptotagmin-1 to Cis or Trans Membranes

Abstract

Neurotransmission involves Ca2+ dependent fusion of synaptic vesicles (SV) to the presynaptic plasma membrane (PM), thereby releasing neurotransmitter into the synaptic cleft. Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARES) drive membrane fusion, but SNAREs are not calcium sensitive. The Ca2+ sensor, Synaptotagmin-1 (Syt1), thus couples neuron depolarization to synchronous neurotransmitter release. Syt1 is a vesicular-tethered protein, with two homologous C2A and C2B domains attached through a juxtamembrane linker. Upon Ca2+ influx, loops on the two domains bind Ca2+ and insert into charged membrane. The C2B domain contains other regions capable of membrane interaction, including the lysine rich polybasic face. Syt1 can interact with either bilayer surface, binding cis- to the SV membrane, or trans- to the PM. Cis-binding likely plays an inhibitory role by back-binding both domains to the SV preventing membrane fusion. Trans-binding then permits fusion, docking the SV to the PM surface. These membrane interactions are lipid specific in order to drive membrane bridging. On the synaptic vesicle surface, only phosphatidylserine (PS) contributes negative charge, while on the PM both PS and phosphatidylinositol-4,5-bisphosphate (PIP2) contribute to the charge density. Through various EPR techniques, we explored cis- and trans-binding under various physiologically relevant lipid, salt, and ionic compositions, to determine if Syt1 may act as a distance regulator between the SV and PM. A series of methods were developed to determine and differentiate membrane insertion of the domains in the full-length protein. With this, we characterized an ATP and PIP2 competition to the polybasic face which blocks or promotes trans-binding. We also characterized the juxtamembrane linker and the preferential binding of C2A to PS and C2B's to PIP2 in the presence of SV or PM mimicking membranes with and without Ca2+.