Abstract
Synaptotagmin I (Syt I) is a vesicle-localized protein implicated in sensing the calcium influx that triggers fast synchronous release of neurotransmitter. How Syt I utilizes its two C2 domains to integrate signals and mediate neurotransmission has continued to be a controversial area of research, though prevalent hypotheses favor independent function. Using differential scanning calorimetry and fluorescence lifetime spectroscopy in a thermodynamic denaturation approach, we tested an alternative hypothesis in which both domains interact to cooperatively disseminate binding information. The free energy of stability was determined for C2A, C2B, and C2AB constructs by globally fitting both methods to a two-state model of unfolding. By comparing the additive free energies of C2A and C2B with C2AB, we identified a negative coupling interaction between the C2 domains of Syt I. This interaction not only provides a mechanistic means for propagating signals, but also a possible means for coordinating the molecular events of neurotransmission.
Highlights
Regulated exocytosis of neurotransmitter requires the fusion of synaptic vesicles with the plasma membrane of the presynaptic neuron
Flexible To determine the relative stability of one domain compared to the other and whether or not the C2 domains of Synaptotagmin I (Syt I) interact, the thermodynamic parameters of C2A and C2B in isolation were extracted from each domain’s thermal denaturation
The two domains differ from one another in extent of reversible folding, with C2B being minimally reversible (5–30%) as assessed by fluorescence lifetime spectroscopy (FLT) as compared to C2A which exhibits nearly complete reversibility
Summary
Regulated exocytosis of neurotransmitter requires the fusion of synaptic vesicles with the plasma membrane of the presynaptic neuron This complex process is mediated by several key proteins including synaptobrevin, syntaxin-1, SNAP-25, complexins, and synaptotagmin I [1,2,3,4,5]. Syt I consists of a short luminal N-terminus, a transmembrane region, and two cytosolic C2 domains in tandem known as C2A and C2B. Both domains bind Ca2+ and acidic phospholipids [9,10,11,12] like phosphatidylserine (PS) and phosphatidylinositol (PIP2), two lipids that modulate fusion [13]. In addition to binding Ca2+ and lipid, Syt I interacts with several proteins involved in vesicle fusion, including members of the SNARE complex [14,15,16,17]
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