Structural basis for the clamping and Ca2+ activation of SNARE-mediated fusion by synaptotagmin

Abstract

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca 2+ ) influx. Here we report the cryo-electron microscopy structure of the Syt1–SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg 2+ )-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the ‘primary’ interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1–SNARE organization is disrupted upon Ca 2+ -influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg 2+ /Ca 2+ ) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1.

Arrangements of proteins at reconstituted synaptic vesicle fusion sites depend on membrane separation

“…Ordered oligomeric rings formed by the C2B domains of Syt1 molecules varying in diameter from 18 to 43 nm (average diameter of 28 nm) have been observed in recent in vitro structural studies [41,58]. The C2B domains partially insert into phospholipid membranes and interact with t‐SNARE helices via the so‐called primary interface [35,39]. In addition, the C2B domains of other Syt1 molecules interact with the opposite side of the SNAREpin via a tripartite interface also containing CpxI [40].…”

“…Such binding blocks the C‐terminal folding of the trans‐SNARE complex, holding it in a partially folded clamped‐like state, preventing membrane fusion. Syt1 also contributes to the inhibitory clamp through interactions between its C2B domain, the trans‐SNARE and Cpx [35]. In this way, the synaptic vesicle is trapped in a docked and primed state on the plasma membrane [35].…”

“…Syt1 also contributes to the inhibitory clamp through interactions between its C2B domain, the trans‐SNARE and Cpx [35]. In this way, the synaptic vesicle is trapped in a docked and primed state on the plasma membrane [35]. Through a poorly understood mechanism, during Ca 2+ influx the C2 domains of Syt1 bind Ca 2+ and anionic lipids causing conformational changes that allow the complete folding/zippering of the trans‐SNARE complex to bring the two membranes into close proximity and subsequently drive their fusion [35–38].…”

“…Crystal structures have revealed two possible Syt1 binding sites on either side of prefusion SNARE–Cpx complexes [39,40] and cryoelectron microscopy (cryo‐EM) studies have shown that Syt1 oligomerises into rings when added to monolayers [41]. These observations led to the proposal of a ‘buttress ring’ model for synaptic protein organisation in which SNAREpins are bound to the plasma membranes via a Syt1 oligomeric ring, and are simultaneously flanked by Syt1 monomers on their vesicle‐facing side [35,42,43]. Furthermore, a recent cellular cryoelectron tomography (cryo‐ET) study suggested a sixfold symmetrical arrangement of synaptic protein complexes in this primed prefusion vesicle state [44].…”

Inhibitory synaptic vesicles have unique dynamics and exocytosis properties

“…The systematic analysis of the components and molecular events of vesicle fusion and neurotransmitter release has generated general models for the organization and functional operation of the pre-and postsynaptic components of synapses (9)(10)(11). The high local Ca 2+ concentration due to activity-induced activation of Ca 2+ channels triggers the local buckling of the plasma membrane through the direct interaction between the C2B domain of synaptotagmin-1 (Syt1) and the lipid on the membrane (12)(13)(14). This leads to synchronous fusion of the membrane and the release of its content into the synaptic cleft (15).…”