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. 2020 Sep 15;119(6):1255-1265.
doi: 10.1016/j.bpj.2020.08.008. Epub 2020 Aug 14.

Membrane Association and Functional Mechanism of Synaptotagmin-1 in Triggering Vesicle Fusion

Ramesh Prasad  1 , Huan-Xiang Zhou  2
Affiliations

Membrane Association and Functional Mechanism of Synaptotagmin-1 in Triggering Vesicle Fusion

Ramesh Prasad et al. Biophys J. .

Abstract

Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2 domains (C2A and C2B). This study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. Although C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently, C2B readily associates with PM via a polybasic cluster and a Ca2+-binding loop. The resulting mechanistic model for the triggering of membrane fusion by synaptotagmin-1 reconciles many experimental observations.

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Figures

Figure 1
Figure 1
Sequence and domain structures of Syt1. (a) Syt1 domain decomposition, containing a transmembrane helix (TM), a disordered linker (LK), and two C2 domains (C2A and C2B). LK contains a basic motif and an acidic motif (underlined sequence). (b) Positioning of the domains of Ca2+-bound Syt1 relative to VM. Selected acidic and basic side chains in LK, C2A, and C2B are shown as sticks; bound Ca2+ ions are shown as spheres. (c) Enlarged view of C2A, highlighting the two Ca2+-binding loops (loops 1 and 3, with the backbone in dark gray and yellow, respectively). Aspartates coordinating Ca2+ ions and basic residues in loop 1, loop 3, loop 2 (backbone in light gray), and the polybasic cluster (backbone in blue) are shown as sticks. (d) Corresponding drawing for C2B. In addition to those with counterparts in C2A, the RR motif is shown with both the backbone and side chains in blue. To see this figure in color, go online.
Figure 2
Figure 2
VM association of LK. (a) Z̄Cα-P, the average distance of each Cα atom from the phosphate plane. Shaded bands represent standard deviations among four replicate simulations. (b) The average number of hydrogen bonds between a linker residue and lipids. The lysines and glutamates near the linker termini are indicated by blue and red arrows, respectively. To see this figure in color, go online.
Figure 3
Figure 3
VM-associated configurations of C2A and C2B in FL simulations. (a) A snapshot from the FL-VM simulations. (b) Illustration of the smallest displacement (Z) of any Cα atom in a C2 domain from the phosphate plane and the tilt angle (Θ) of the domain. (c) Scatter plot of C2A Θ and Z collected from the simulations. The red and blue ovals indicate ensembles in the tilted and straight poses, respectively. The histogram in Θ was fitted to a sum of two Gaussians, corresponding to the two types of poses. (d) Left: enlarged view of C2A in the tilted pose shown in (a). Right: C2A in the straight pose from a different snapshot. R233 in loop 3 is labeled. (e) Scatter plot of C2B Θ and Z. Red and black dots represent VM-associated and -dissociated snapshots, respectively; red and blue ovals indicate ensembles in the tilted and sideways poses, respectively, of VM-associated C2B. The Θ histogram of VM-associated C2B was fitted to a sum of two Gaussians. (f) Right: enlarged view of C2B in the sideways pose shown in (a). Left: C2B in the tilted pose from a different snapshot. K366 in loop 3 is labeled. To see this figure in color, go online.
Figure 4
Figure 4
Comparison of VM and PM association of C2AB. (a) Fractions of snapshots in which individual C2AB residues form membrane contacts in the FL-VM simulations. The sums of membrane contact fractions of residues in some motifs (e.g., loop 3) are indicated. (b) Counterparts for the C2AB-PM simulations. (c) Scatter plot of C2A Θ and Z collected from the C2AB-PM simulations. (d) Scatter plot of C2B Θ and Z. (e) A snapshot from the C2AB-PM simulations. (f) Enlarged view of C2A showing PM interactions. (g) Enlarged view of C2B. To see this figure in color, go online.
Figure 5
Figure 5
Interactions of the linker acidic motif with C2B Ca2+-binding loops. (a) Snapshot at 193 ns of FL-VM sim1. Shown on the right is an enlarged view that illustrates the interactions of Glu131, Glu132, and Glu134 interacting with loops 1 and 3 and a Ca2+ ion in C2B. (b) The fractions of snapshots in which individual C2B residues form contacts with acidic residues in the linker, among all snapshots in which at least one such contact is formed. To see this figure in color, go online.
Figure 6
Figure 6
PM association of C2B released from VM. (a) An initial snapshot in which C2B released from VM is placed near PM. (b) Time traces of C2B Θ and Z. (c) Time trace of C2B contacts with PIP2 molecules or with all PM lipids; each trace is smoothed using the running average in a 11-ns window. Horizontal dashed lines indicate the average C2B-PIP2 contact numbers from 50 to 250 ns and from 300 to 700 ns. (d) A snapshot in which C2B becomes stably associated with PM, showing the polybasic cluster and loop 3 (side chains shown as sticks) interacting with PIP2 molecules (shown as the red surface). To see this figure in color, go online.
Figure 7
Figure 7
For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.bpj.2020.08.008. Proposed model for the triggering of membrane fusion by Syt1. By interacting with the Ca2+-binding loops, the linker acidic motif flips C2B from VM-facing to PM-facing. C2B then quickly associates with PM, providing a platform for the complete assembly of the trans-SNARE complex. To see this figure in color, go online.
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