Synaptotagmin-1 C2B domains cooperatively stabilize the fusion stalk via a master-servant mechanism

Abstract

Synaptotagmin-1 is a low-affinity Ca²⁺ sensor that triggers synchronous vesicle fusion. It contains two similar C2 domains (C2A and C2B) that cooperate in membrane binding, being the C2B domain mainly responsible for the membrane fusion process due to its polybasic patch KRLKKKKTTIKK (321-332). In this work, a master-servant mechanism between two identical C2B domains is shown to control the formation of the fusion stalk in a calcium-independent manner. Two regions in C2B are essential for the process, the well-known polybasic patch and a recently described pair of arginines (398 399). The master domain shows strong PIP₂ interactions with its polybasic patch and its pair of arginines. At the same time, the servant analogously cooperates with the master to reduce the total work to form the fusion stalk. The strategic mutation (T328E, T329E) in both master and servant domains disrupts the cooperative mechanism, drastically increasing the free energy needed to induce the fusion stalk, however, with negligible effects on the master domain interactions with PIP₂. These data point to a difference in the behavior of the servant domain, which is unable to sustain its PIP₂ interactions neither through its polybasic patch nor through its pair of arginines, and in the end, losing its ability to assist the master in the formation of the fusion stalk.

Fig. 1. Schematics of the fusion stalk. (a) Organelles about to fuse in the cytosol. (b) Formation of the fusion stalk while interacting with Syt1–C2B domains. Arginines R398 and R399 and the polybasic region 321–332 are highlighted in vdW representations.

Fig. 2. Membrane-only fusion stalk formation. (a) PMF for membrane-only and forward and backward directions. Error bars are standard errors calculated by individually splitting the profiles in independent blocks. (b) Unbiased molecular dynamics showing the local minimum at ξ_f ∼0.55. Each circle represents an inter-membrane lipid count value for a single configuration, and the color represents its occurrence, from low (blue) to high (red). (c) Molecular dynamics snapshots showing the formation of the stalk. Lipid molecules (top) are shown in grey with PO₄ beads in yellow, while water molecules (bottom) are blue surfaces.

Fig. 3. Free energy profiles for the fusion stalk. The membrane-only system (black line) is taken as the reference. Repetitions under identical conditions for the same bilayers now containing: one Syt1–C2B wild-type domain (violet line), two wild-type C2B domains (blue line) and two mutant T328E and T329E C2B domains (green line). Error bars are standard errors calculated by individually splitting the profiles in independent blocks.

Fig. 4. Fusion stalk evolution. (a) Inter-membrane lipid count along the evolution of the collective variable ξ_f for three cases: membrane-only (gray circles), 2 Syt1–C2B wild-type domains (blue squares) and 2 Syt1–C2B mutant domains (green diamonds). Also, piece-wise interpolating curves are superimposed for each group of data. The inset shows the region where the first stalk forms. (b) Normalized heat-map colored densities for tail beads only, showing the configurational transformation of the toroid for the membrane-only system, as shown in the scheme below.

Fig. 5. Master-servant C2B domain orientation during unbiased μs-length molecular dynamics. (a) Distribution of end-to-end Z distances in C2B domains for two independent simulations performed for initially flat and parallel bilayers, the first one with 2 wild-type C2B domains (black and blue histograms) and the second one with 2 mutant T328E and T329E C2B domains (red and green histograms). Ribbon representation of the C2B domain schematizes the change of orientation observed. (b) PO₄:PO₄ inter-membrane distance for three systems: membranes only (black line), bilayers with 2 wild-type C2B domains (blue line) and bilayers with 2 mutant T328E and T329E C2B domains (green line). (c) Averaged densities with POPC in red, POPS in green and PIP₂ in blue. The left panel corresponds to 2 wild-type C2B domains, and the right panel corresponds to 2 mutant C2B domains. C2B domains are yellow. Water molecules are not shown. Data collection from μs-length unbiased molecular dynamics started from planar and parallel bilayers.

Fig. 6. The master-servant C2B mechanism. Radial distribution functions of PIP₂ lipids measured from the polybasic region (positions 321–332) and arginines (positions 398 399). In all panels, 2 wild-types C2B domains are black and blue lines, and 2 T328E and T329E mutant domains are red and green lines. (a) RDF for PIP₂ with polybasic regions in master proteins (1 wild-type and 1 mutant) as the reference. (b) RDF for PIP₂ with polybasic regions in servant proteins (1 wild-type and 1 mutant) as the reference. (c) RDF for PIP₂ with arginines R398 and R399 in master proteins (1 wild-type and 1 mutant) as the reference. (d) RDF for PIP₂ with arginines R398 and R399 in servant proteins (1 wild-type and 1 mutant) as the reference. Data collected from μs-length unbiased molecular dynamics started at ξ_f ∼0.85.