Analysis of SNARE complex/synaptotagmin-1 interactions by one-dimensional NMR spectroscopy

Neurotransmitter release depends critically on the Ca²⁺ sensor synaptotagmin-1 and the SNARE proteins syntaxin-1, synaptobrevin, and SNAP-25, which mediate membrane fusion by forming tight SNARE complexes that bridge the synaptic vesicle and plasma membranes. Interactions between the SNARE complex and the two C₂ domains of synaptotagmin-1 (the C₂A and C₂B domains) are believed to play a key role in coupling Ca ²⁺ sensing to membrane fusion, but the nature of these interactions is unclear, in part because of a paucity of data obtained by quantitative biophysical methods. Here we have analyzed synaptotagmin-1/SNARE complex interactions by monitoring the decrease in the intensities of one-dimensional ¹³C-edited ¹H NMR spectra of ¹³C-labeled fragments of synaptotagmin-1 upon binding to unlabeled SNARE complex. Our results indicate that there is a primary binding mode between synaptotagmin-1 and the SNARE complex that involves a polybasic region in the C₂B domain and has a sub-micromolar affinity. Our NMR data, combined with precipitation assays, show that there are additional SNARE complex/ synaptotagmin-1 interactions that lead to aggregation and that involve in part two arginines at the bottom of the C₂B domain. Overall, this study shows the importance of disentangling the contributions of different types of interactions to SNARE complex/synaptotagmin-1 binding and illustrates the usefulness of one-dimensional NMR methods to analyze intricate protein interactions.