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

Amy Zhou, Kyle D. Brewer, Jose Rizo-Rey

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3446-3456
Number of pages11
JournalBiochemistry
Volume52
Issue number20
DOIs
StatePublished - May 21 2013

Fingerprint

Synaptotagmin I
SNARE Proteins
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Membrane Fusion
Nuclear magnetic resonance
Fusion reactions
Syntaxin 1
R-SNARE Proteins
Membranes
Synaptic Membranes
Synaptic Vesicles
Cell membranes
Neurotransmitter Agents
Arginine
Assays
Agglomeration
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Analysis of SNARE complex/synaptotagmin-1 interactions by one-dimensional NMR spectroscopy. / Zhou, Amy; Brewer, Kyle D.; Rizo-Rey, Jose.

In: Biochemistry, Vol. 52, No. 20, 21.05.2013, p. 3446-3456.

Research output: Contribution to journalArticle

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