Rearrangements under confinement lead to increased binding energy of Synaptotagmin-1 with anionic membranes in Mg²⁺...

Synaptotagmin-1 (Syt1) is the primary calcium sensor (Ca²⁺ ) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca²⁺ -dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane-anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 k_B T in EGTA, ~10 k_B T in Mg²⁺ and ~18 k_B T in Ca²⁺ . Molecular rearrangements measured during confinement are more prevalent in Ca²⁺ and Mg²⁺ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca²⁺ -activation process in synaptic transmission.