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

Abstract

Neuronal 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 Ca 2+ 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. AbstractRelease of neuronal transmitters from nerve terminals is triggered by the molecular Ca 2+ 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 Ca 2+ 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. 4The palmitoyl oleyl phosphatidylcholine (POPC) lipid bilayers were generated using VMD. The initial structure of anionic lipid bilayer containing phosphatidylserine (POPS) and PIP2 , POPC:POPS:PIP2 (75:20:5) [74] was kindly provided by Dr. J. Wereszczynski (Illinois Institute of Technology). In all the systems, the lipid bilayers were positioned in the XY plain.The initial structures for the isolated C2A [75]) and C2B [76] domains in their Ca 2+ -bound forms were obtained from crystallography studies (1BYN and 1TJX, respectively in the Protein Data Base). The initial structures of the isol...

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

“…Previous work showed calcium binding to isolated Syt1 C2 domains leads to insertion of the hydrophobic residues at the tips of both of the the calcium-binding loops into the membrane (41, 96, 104, 125) (however, see ref. (126)). In the presence of PI(4,5)P 2 , calcium-bound C2B assumes a conformation in which its long axis is tilted with respect to the membrane normal, as it interacts with the membrane simultaneously through its calcium binding loops and the polybasic patch (K324-327) bound to PI(4,5)P 2 (95, 127).…”

All-atom molecular dynamics simulations of synaptic vesicle fusion I: a glimpse at the primed state

“…Although continuous helices were observed in the crystal structure of a cis-SNARE complex that represents the configuration occurring after membrane fusion (Stein et al, 2009), the natural expectation is that the helical structure must break somewhere to accommodate the geometry of a trans-SNARE complex, most likely at the juxtamembrane linker. This expectation has been supported experimentally (Kim et al, 2002) and with all-atom MD simulations (Bykhovskaia, 2021). Moreover, helix continuity in the linkers is not required for neurotransmitter release (Kesavan et al, 2007; Zhou et al, 2013).…”

“…Although the time scales reachable with all-atom MD simulations are more limited than those attainable with coarse-grained simulations, high performance computing currently allows calculation of microsecond trajectories for systems containing millions of atoms. Indeed, all-atom simulations have already provided important insights into interactions among the components of the primed complex (Bykhovskaia, 2021; Bykhovskaia et al, 2013). Note also that the delay from Ca 2+ influx into the presynaptic terminal to observation of postsynaptic currents in rat cerebellar synapses at 38°C is 60 μs (Sabatini and Regehr, 1996), and that multiple events occur within this time frame, including Ca 2+ binding to the sensor, Ca 2+ -evoked synaptic vesicle fusion, opening of the fusion pore, diffusion of neurotransmitters through the synaptic cleft, binding of the neurotransmitters to their postsynaptic receptors and opening of the channels that underlie the postsynaptic currents.…”

Ferlins and TgDOC2 in Toxoplasma Microneme, Rhoptry and Dense Granule Secretion

“…In general, the absolute requirement for SNAREs in exocytosis, certainly regarding ferlin-mediated exocytosis, has come under debate. It has been postulated that fast, Ca 2+ -dependent exocytosis is inconsistent with the role of SNAREs [ 104 ] and some exocytosis in absence of SNAREs is possible [ 21 , 105 ]. Furthermore, some neurotransmitter is still released when all relevant SNAREs are depleted [ 105 ], and alternative models for SNARE independent neurotransmitter release have been postulated [ 104 ].…”

“…C2 domains are composed of 80–160 amino-acids that fold into β-sheets with specific binding characteristics that are found in a large and diverse set of eukaryotic proteins that can be grouped into several families [ 20 ]. Most C2 domains have lipid binding characteristics that are family specific [ 21 ], but they can also interact with other proteins [ 22 , 23 , 24 ]. The most diverse C2 domain protein family is derived from a protein kinase C (PKC) archetype that is unique among C2 domains in that it can stabilize Ca 2+ ions (up to 3 per C2 domain) in a pocket composed of three loops emanating from the β-sheets.…”