Studies on the Structure and Interactions of Synaptotagmin-1 and SNARE Proteins in Neurotransmitter Release

The SNARE complex and synaptotagmin-1 are essential for Ca²⁺-evoked neurotransmitter release, yet the mechanism of how these proteins work together in membrane fusion is unclear. Dozens of studies performed over two decades have described different types of synaptotagmin-1/SNARE interactions and reported the individual structures of the SNAREs, the SNARE complex, and the C₂ domains that form most of the cytoplasmic region of synaptotagmin-1. However, a high-resolution structure of a synaptotagmin-1/SNARE complex, which is crucial to understand the mechanism of release, has not been reported. In this work, we explore methods to examine the biophysical properties of synaptotagmin-1 and the SNAREs, primarily using NMR. We first examine the conformation of synaptobrevin on nanodisc bilayers and find that that the N-terminal portion of the SNARE domain has a high propensity to remain unfolded on membranes. We next look at the conformation of synaptotagmin-1 on nanodiscs and demonstrate that although both C₂ domains primarily bind to the same membrane, a small population of antiparallel conformers also exist. Finally and most importantly, we look at the structure of synaptotagmin-1/SNARE complex. After overcoming many obstacles and failed approaches, we were able to obtain intermolecular restraints for this 66 kDa machinery by introducing lanthanide tags for measurement of pseudocontact shifts (PCSs). Computational analyses incorporating these restraints show that a static structure cannot fully explain all the PCS measurements, but the data can be fit with a dynamic ensemble of structures whereby a polybasic region of the synaptotagmin-1 C₂B domain binds to a polyacidic region formed by the syntaxin-1 and SNAP-25 SNARE motifs. The orientation of the synaptotagmin-1 C₂B domain with respect to the SNARE complex within the ensemble is expected to allow quick, simultaneous interaction with lipids on both membranes upon Ca²⁺ binding to bring the membranes together. Distinct mutations in the C₂B domain polybasic region caused differential disruptions of SNARE complex-binding that correlate with the impairment of neurotransmitter release caused by these mutations. Overall, these results and the architecture of the synaptagmin-1/SNARE complex revealed by our NMR data support the hypothesis that synaptotagmin-1 cooperates with the SNAREs by bringing membranes together to trigger fast fusion upon Ca²⁺ influx.