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A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release and Studies of Synaptotagmin-1 action by all-atom molecular dynamics simulations

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Dec 03, 2024 version files 31.67 GB

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Abstract

Abstract 1:

Neurotransmitter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2-domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a ‘primary interface’ comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding through the primary interface orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. To clarify this paradox, we have used NMR and fluorescence spectroscopy. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I, and that a mutation that impairs spontaneous and Ca2+-triggered neurotransmitter release enhances binding through region I. Moreover, fluorescence assays show that Ca2+ does not induce dissociation of synaptotagmin-1 from membrane-anchored SNARE complex but causes reorientation of the C2B domain. Based on these results and electrophysiological data described in Toulme et al. (https://doi.org/10.1073/pnas.2409636121), we propose that upon Ca2+ binding the Synaptotagmin-1 C2B domain reorients on the membrane and dissociates from the SNAREs at region I but not region II, acting remotely as a lever that pulls the SNARE complex and facilitates linker zippering or other SNARE structural changes required for fast membrane fusion.

Abstract 2:

Neurotransmitter release is triggered in microseconds by the two C2 domains of the Ca2+ sensor Synaptotagmin-1 and by SNARE complexes, which form four-helix bundles that bridge the vesicle and plasma membranes. The Synaptotagmin-1 C2B domain binds to the SNARE complex via a ‘primary interface’, but the mechanism that couples Ca2+-sensing to membrane fusion is unknown. Widespread models postulate that the Synaptotagmin-1 Ca2+-binding loops accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but these models do not seem compatible with SNARE binding through the primary interface, which orients the Ca2+-binding loops away from the fusion site. To test these models, we performed molecular dynamics simulations of SNARE complexes bridging a vesicle and a flat bilayer, including the Synaptotagmin-1 C2 domains in various configurations. Our data do not support the notion that insertion of the Synaptotagmin-1 Ca2+ binding loops causes substantial membrane curvature or major perturbations of the lipid bilayers that could facilitate membrane fusion. We observed membrane bridging by the Synaptotagmin-1 C2 domains, but such bridging or the presence of the C2 domains near the site of fusion hindered the action of the SNAREs in bringing the membranes together. These results argue against models predicting that Synaptotagmin-1 triggers neurotransmitter release by inducing membrane curvature, perturbing bilayers or bridging membranes. Instead, our data support the hypothesis that binding via the primary interface keeps the Synaptotagmin-1 C2 domains away from the site of fusion, orienting them such that they trigger release through a remote action.