Abstract
Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) 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 (Mg2+)-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 Ca2+-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1.
Highlights
Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx
We used a protein construct wherein the minimal C2AB domains of Syt[1] (Syt1C2AB) are covalently linked to the SNARE complex, which was proven to be successful in 3D crystal formation[41]
Negative stain EM analysis of Syt1C2AB–SNARE protein complex incubated with liposomes containing negatively charged lipids (DOPC/DOPS/ PIP2 60/34/6) revealed the formation of protein-coated lipid tubules with helical diffraction peaks (Supplementary Fig. 1)
Summary
Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx. The C2B domain concurrently interacts the SNARE bundle via the ‘primary’ interface and is positioned between the SNAREpins and the membrane In this configuration, Syt[1] is projected to sterically delay the complete assembly of the associated SNAREpins and contribute to clamping fusion. Synaptotagmin-1 (Syt1) is a key component involved in all stages of the process, including SV docking and priming[5,6,7,8,9], preventing un-initiated SV fusion[10,11] and triggering fusion upon Ca2+ influx[12,13,14,15] Such broad specialization has not resulted in structural complexity of the protein. Recent studies show that the C2A domain can modulate the Syt[1] function[28,29,30,31]
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