Analysis of Synaptotagmin-SNARE Complex Interactions by One-Dimensional NMR Spectroscopy

The mechanism of calcium-triggered neurotransmitter release is mediated by numerous proteins at the neuronal synapse. The SNARE proteins form a complex that mediates fusion between the synaptic vesicle and plasma membrane. The protein synaptotagmin-1 is the major sensor for the calcium concentration. Synaptotagmin-1 and the SNARE complex are therefore believed to interact in order to couple the calcium concentration to membrane fusion. The detailed mechanism of the interaction is still unclear, because the technical difficulties in probing the system have rendered it intractable with respect to traditional biochemical and biophysical methods. I will present an analytical method based on one-dimensional NMR spectroscopy that overcomes these limitations. The experiments are based on the current working model that synaptotagmin-1 facilitates the SNAREs’ role in membrane fusion in a calcium-dependent manner. My data suggest that the synaptotagmin-1/SNARE complex interaction is calcium-dependent and mediated primarily by the synaptotagmin C2B domain. Further, the polybasic region of C2B constitutes the primary binding site, while the two arginine residues at the bottom of the domain mediate additional interactions that lead to aggregation and precipitation. These results help clarify the complex mechanism of synaptotagmin-1/SNARE coupling, as well as to illustrate the usefulness of 1D NMR to study such protein-protein interactions. Alternative methods to probe such interactions are explored. The advantage of a related competition assay lies in its sampling of the primary binding site with little interference of the other binding mode(s). However, experimental artifacts hindered the application of the assay to my system. A diffusion-based method is another route for studying protein-protein interactions, provided there is sufficient dynamic range to allow for meaningful interpretations. The SNARE-, calcium-, and lipid-binding profiles of an extended synaptotagmin-like protein (E-Syt) were also characterized. E-Syts are of interest because they can shed light on the evolution of proteins in the synaptotagmin family. In addition, they can reveal general governing principles of tandem C2-domain proteins which often function in signal transduction and membrane trafficking.