Synaptotagmin signal propagation: a model system for functional cooperativity at the membrane

Abstract

Synaptotagmin I (Syt I) is a snaptic vesicle integral membrane protein that has been genetically and biochemically identified as the calcium sensor for synchronous release of neurotransmitter. How Syt I senses a calcium influx and subsequently conveys the information to its numerous binding partners to coordinate neurotransmission has been a question of extensive study for decades; however, an underlying mechanism for this function has yet to be determined. To address this issue, we hypothesized that Syt I's signaling capabilities stem from marginal stability of and intrinsic disorder within its protein structure. Both features can provide great signaling capacity and structure plasticity required for the protein's diverse and responsive functionalities observed in vivo. To test this hypothesis, a combination of differential scanning calorimetry, isothermal titration calorimetry, and fluorescence lifetime spectroscopy were used to determine the stability and binding for Syt I's cytosolic region in buffered solution. First, to test for cooperative signaling between Syt I's tandem C2 domain (C2A and C2B), both domains were thermally denatured separately and tethered together. By comparing the very weak, but additive free energies of these three protein constructs, it was found that the two marginally stable C2 domains cooperatively interact through negative coupling, a destabilizing interaction. Second, to test the potential impact of disorder on Syt I's calcium sensing function, two constructs of the C2A domain (one with the long disordered C2 domain-membrane linker and one without) were thermally denatured and titrated with saturating calcium. The C2A construct with the disordered linker was found to be less stable and bound calcium in a cooperative manner. The C2A construct without the disordered linker was more stable and did not exhibit cooperative binding. Taken together, the above findings support the hypothesis that Syt I's cooperative signaling likely stem from weak energetics. Such a model for protein function suggests Syt I coordinates and integrates its interactions through subtle refinements in its conformational ensemble.