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. 2013 May 21;52(20):3446-56.
doi: 10.1021/bi400230u. Epub 2013 May 7.

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

Amy Zhou  1 , Kyle D Brewer, Josep Rizo
Affiliations

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

Amy Zhou et al. Biochemistry. .

Abstract

Neurotransmitter release depends critically on the Ca(2+) 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 (13)C-edited (1)H NMR spectra of (13)C-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.

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Figures

Figure 1
Figure 1
Ribbon diagrams of the synaptotagmin-1 C2A and C2B domains. Ca2+ ions are shown as green spheres. The side chains that were mutated are represented by stick models and labeled.
Figure 2
Figure 2
A. Expansions of the region containing the SMR of 1D 13C-edited 1H-NMR spectra of 3 µM 15N,13C-labeled C2AB fragment acquired in the presence of 1 mM EDTA (-Ca2+) or 1 mM Ca2+ (+Ca2+), and in the absence (−SC) or presence (+SC) of 3.5 µM SNARE complex. B. analogous expansions of 1D 13C-edited 1H-NMR spectra of 3 µM 15N,13C-labeled C2AB fragment in the presence of 3.5 µM SNARE complex and the indicated concentrations of Ca2+ in µM units. C. Plot of the normalized SMR intensities observed in 1D 13C-edited 1H-NMR spectra of 3 µM 15N,13C-labeled C2AB fragment in the presence of 3.5 µM SNARE complex and variable concentrations of Ca2+. A subset of the data corresponding to this titration is shown in panel B. The curve shows the fit to a Hill equation.
Figure 3
Figure 3
Titrations of WT and mutant 15N,13C-labeled C2AB fragments with SNARE complex monitored by 1D 13C-edited 1H-NMR spectra. A. Plot of the SMR intensities observed in 1D 13C-edited 1H-NMR spectra of 3 µM WT C2AB fragment as a function of unlabeled SNARE complex concentration in the presence of 1 mM Ca2+. B-D. Plots for analogous titrations performed with DN (B), RR (C) and KK (D) mutant C2AB fragments (open squares), superimposed with a plot obtained for the WT C2AB fragment (closed circles; the same shown in A). The curves show the fits of the data obtained with equation (1).
Figure 4
Figure 4
Expansions showing the methyl region of 1D 13C-edited 1H-NMR spectra of 3 µM WT (A) or RR mutant (B) C2AB fragment in 1 mM Ca2+ and in the absence or presence of 20 µM SNARE complex (SC).
Figure 5
Figure 5
Analysis of the relative contributions of the synaptotagmin-1 C2 domains to SNARE complex binding. A. Plots of the SMR intensities observed in 1D 13C-edited 1H-NMR spectra of 3 µM synaptotagmin-1 C2A domain (solid squares), pure C2B domain (solid cirlces) and impure C2B domain (open squares) as a function of unlabeled SNARE complex concentration in the presence of 1 mM Ca2+. B. 1H-15N HSQC spectra of synaptotagmin-1 C2B domain that was fully purified (red contours) or that was purified by our standard protocol but omitting the final cation exchange chromatography (black contours). The insets show expansions of regions containing cross-peaks that are unique for the purified C2B domain but exhibit multiplicity in the impure C2B domain [see ref. (49)].
Figure 6
Figure 6
Titrations of WT and mutant 15N,13C-labeled C2B domain with SNARE complex monitored by 1D 13C-edited 1H-NMR spectra. A–B. Plots of the SMR intensities observed in 1D 13C-edited 1H-NMR spectra of 3 µM 15N,13C-labeled RR (A) or KK (B) mutant C2B domain as a function of unlabeled SNARE complex concentration in the presence of 1 mM Ca2+ (open squares), superimposed with a plot obtained for the WT C2B domain (closed circles). The curves show the fits of the data obtained with equation (1).
Figure 7
Figure 7
Expansions showing the methyl region of 1D 13C-edited 1H-NMR spectra of 3 µM WT (A) or RR mutant (B) C2B domain in 1 mM Ca2+ and in the absence or presence of 20 µM SNARE complex (SC).
Figure 8
Figure 8
Analysis of the solubility of WT and mutant synaptotagmin-1 fragments in the presence of SNARE complex. Samples containing 10 µM C2AB fragment (A,C) or 10 µM C2B domain (B,D) were incubated with 10 µM (A,B) or 20 µM (C,D) SNARE complex for 5 min in the presence of 1 mM Ca2+. The soluble fractions (S) and the precipitates (P) were separated by centrifugation and analyzed by SDS PAGE followed by Coomassie blue staining. The positions of the C2AB fragment (C2AB), C2B domain (C2B) and SNARE complex (SC) are indicated. Note that the C2AB fragment runs above the SNARE complex (panels A,C) but the C2B domain runs below the SNARE complex (panels B,D) because of its smaller size. Note also that the KK and DN mutant C2AB fragments run slightly differently than the WT C2AB fragment, which most likely arises because the mutations change charged residues.
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