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

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.