Analysis of SNARE Complex/Synaptotagmin-1 Interactions by One-Dimensional NMR Spectroscopy

作者：Zhou Amy; Brewer Kyle D; Rizo Josep^*

来源：Biochemistry, 2013, 52(20): 3446-3456.

DOI：10.1021/bi400230u

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 C-2 domains of synaptotagmin-1 (the C(2)A and C2B domains) are believed to play a key role in coupling Ca2+ 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 C-13-edited NMR spectra of C-13-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-rnicromolar 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.

• 出版日期2013-5-21