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 the main 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. 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, in the end losing its ability to assist the master in the formation of the fusion stalk.

Enhanced Expansion and Reduced Kiss-and-Run in Fusion Pores Steered by Synaptotagmin-1 C2B Domains

“…Based on an event-oriented classification for the different stages of the fusion pore 18,23,[33][34][35] and in resemblance with the biological process of a vesicle about to fuse with the plasma membrane, as schematically shown in figs. 1a and 1b, we have used independent collective variables for membrane fusion (ξ f ), 28 fusion pore nucleation (ξ n ) and expansion (ξ e ), see eqs. 1, 2 and 3.…”

“…The collective variables nomenclature follows the same formulation originally developed by Hub and collaborators 19,25,26 and our previous works in membrane fusion 28 and fusion pore nucleation. 24 Conceptually, ξ f and ξ n are analogous (see eqs.…”

“…1 to fuse membranes inducing a fusion stalk. 28 Here, starting from the fusion stalk we have sequentially used eq. 2 for fusion pore nucleation and eq.…”

“…Together with our previous studies on membrane fusion and pore nucleation, 24,28 we complete here another step along the life of the fusion pore by describing its expansion. We have ported the nucleation and expansion processes into two convenient collective variables for the PLUMED 2 development version environment (https://github.com/plumed/plumed2), 29 easily compilable as an optional module and with source codes freely available on GitHub: https://github.com/lautarodibartolo/FusionPore.…”

“…Starting from a fusion stalk 28 as the initial configuration, we have nucleated a fusion pore using eq. 2.…”