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

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.