Biology subjects

Bykhovskaia, M.

Publications and source records attributed to Bykhovskaia, M..

4 recordsLinked to original sources

bioRxiv2026

Disease-causing mutations in Synaptotagmin can act via dominant-negative, gain-of-function or haploinsufficient mechanisms

Members of the Synaptotagmin (SYT) family of synaptic vesicle (SV) proteins contain two Ca2+ binding C2 domains (C2A and C2B) that regulate the timing and probability of SV fusion. Recently, dominant mutations in SYT1, present at most CNS synapses, and SYT2, abundant in the PNS, have been found to cause human neurological diseases. Most pathogenic alleles localize to the C2B Ca2+ binding pocket, although mutations outside of this region have been identified. Here we report the effects of a large array of disease-associated human SYT point mutations on synaptic transmission using the Drosophila model to define their mechanism of action. Mutations in the C2B Ca2+ binding pocket were the most severe and acted in a dominant-negative manner to decrease evoked release. Indeed, mutations in the C2B Ca2+ binding pocket were a privileged site for dominant-negative effects on SV fusion. Molecular dynamics simulations indicate these mutations alter the topology of the C2B Ca2+ binding pocket and disrupt Ca2+-triggered membrane insertion. Several mutations outside of this region acted through a gain-of-function mechanism to enhance neurotransmitter release, while others caused the protein to undergo degradation. Consistent with this latter subset acting via haploinsufficiency, heterozygotes lacking one copy of SYT1 displayed a [~]40% decrease in evoked release. These data indicate C2B Ca2+ binding pocket mutations act dominantly to poison the fusion machinery and block release sites, while mutations outside of this region cause haploinsufficiency or gain-of-function effects with milder effects on synaptic output and behavioral phenotypes.

neuroscience↗

bioRxiv2024

Dynamic Formation of the Protein-Lipid Pre-fusion State

Synaptic vesicles (SVs) fuse with the presynaptic membrane (PM) to release neuronal transmitters. The SV protein Synaptotagmin 1 (Syt1) serves as a Ca2+ sensor for evoked fusion. Syt1 is thought to trigger fusion by penetrating into PM upon Ca2+ binding, however the mechanistic detail of this process is still debated. Syt1 interacts with the SNARE complex, a coiled-coil four-helical bundle that enables the SV-PM attachment. The SNARE-associated protein Complexin (Cpx) promotes the Ca2+-dependent fusion, possibly interacting with Syt1. We employed all-atom molecular dynamics (MD) to investigate the formation of the Syt1-SNARE-Cpx complex interacting with the lipid bilayers of PM and SV. Our simulations demonstrated that the PM-Syt1-SNARE-Cpx complex can transition to a "dead-end" state, wherein Syt1 attaches tightly to PM but does not immerse into it, as opposed to a pre-fusion state, which has the tips of the Ca2+-bound C2 domains of Syt1 inserted into PM. Our simulations unraveled the sequence of Syt1 conformational transitions, including the simultaneous Syt1 docking to the SNARE-Cpx bundle and PM, followed by the Ca2+ chelation and the penetration of the tips of Syt1 domains into PM, leading to the pre-fusion state of the protein-lipid complex. Importantly, we found that the direct Syt1-Cpx interactions are required to promote these transitions. Thus, we developed the all-atom dynamic model of the conformational transitions that lead to the formation of the pre-fusion PM-Syt1-SNARE-Cpx complex. Our simulations also revealed an alternative "dead-end" state of the protein-lipid complex that can be formed if this pathway is disrupted. Statement of SignificanceNeurons communicate by releasing transmitter molecules. Transmitters are packed in synaptic vesicles (SVs) and released by the fusion of SVs with the presynaptic membrane (PM). This process is regulated by a dynamic complex of fusion proteins, including the coil-coiled SNARE bundle that attaches SV to PM, Synaptotagmin that serves as a Ca2+ sensor for the release process, and Complexin that attaches to the SNARE bundle and promotes the Ca2+-dependent release. To understand how these proteins interact dynamically with the lipid bilayers of SV and PM, we employed molecular dynamics, a computational approach that enables simulating the behavior of proteins and lipids at the atomistic resolution. Our simulations enabled us to delineate the stages of the formation of prefusion protein-lipid complex.

biophysics↗

bioRxiv2020

SNARE Complex Alters the Interactions of the Ca2+ sensor Synaptotagmin 1 with Lipid Bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca2+ sensor Synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca2+ binding. Prior to fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which binds the C2B domain of Syt1. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. The MD simulations showed that the C2AB tandem of Syt1 can either bridge SV and PM or immerse into PM, and that the latter configuration is more favorable energetically. Surprisingly, C2 domains did not cooperate in penetrating into PM, but instead mutually hindered the lipid penetration. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the Syt1-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains of Syt1 and promotes the immersion of both domains into the PM bilayer. Subsequently, we simulated this pre-fusion protein complex between lipid bilayers imitating PM and SV and found that the immersion of Syt1 into the PM bilayer within this complex promotes PM curvature and leads to lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex. Statement of SignificanceNeuronal transmitters are packed in synaptic vesicles (SVs) and released by fusion of SVs with the presynaptic membrane (PM). SVs are attached to PM by the SNARE protein complex, and fusion is triggered by the Ca2+ sensor Synaptotagmin 1 (Syt1). Although Syt1 and SNARE proteins have been extensively studied, it is not yet fully understood how the interactions of Syt1 with lipids and the SNARE complex induce fusion. To address this fundamental problem, we took advantage of Anton2 supercomputer, a unique computational environment, which enables simulating the dynamics of molecular systems at a scale of microseconds. Our simulations produced a dynamic all-atom model of the prefusion protein-lipid complex and demonstrated in silico how the Syt1-SNARE complex triggers fusion.

biophysics↗

bioRxiv2018

Coarse-Grained Model of the SNARE Complex Shows that Quick Zippering Requires Partial Assembly

Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the presynaptic membrane. Vesicles become attached to the membrane via the SNARE complex. The SNARE complex comprises the vesicle associate protein Synaptobrevin (Syb), the membrane associated protein syntaxin (Syx), and the cytosolic protein SNAP25, which together form a four-helical bundle. The full assembly of Syb onto the core SNARE bundle promotes vesicle fusion. We investigated SNARE assembly using a coarse-grained model of the SNARE complex. The model retains chemical specificity, and was calibrated using single molecule experiments and all-atom molecular dynamics simulations. Steered force-control simulations of SNARE unzippering by peeling off Syb were used to set up initial disassembled states of the SNARE complex. From these states, the assembly process was simulated. We found that if Syb is in helical form, then the SNARE complex assembles rapidly, on a sub-microsecond time-scale. We found that assembly times grow exponentially with separation distance between Syb and Syx C-termni. The formation of helical turns is likely to substantially decelerate the assembly, consistent with single molecule force experiments that show SNARE assembly duration on the time-scale of hundreds of ms. Since synaptic vesicle fusion occurs at a sub-millisecond time-scale, our results indicate that for biologically relevant rapid assembly the SNARE complex needs to be partially zippered and its constituent helices brought into proximity, possibly by means of molecular chaperones.

biophysics↗