1. Volume 24, Issue 2, Pages 363-376 (October 1999)
Kinetics of Synaptotagmin Responses to Ca2+ and Assembly with the Core SNARE Complex onto Membranes 
Anson F Davis, Jihong Bai, Dirk Fasshauer, Mark J Wolowick, Jessica L Lewis, Edwin R Chapman 
Neuron 
Volume 24, Issue 2, Pages (October 1999)
DOI: /S (00)

2. Figure 5
Sequential Insertion of Loops 1 and 3 of C2A into Lipid Bilayers
(A) Ca2+ binding–loops 1 and 3 of C2A penetrate into lipid bilayers. Upper panel corresponds to C2A-F234W, lower panel corresponds to C2A-M173W. Protein (10 μM) was excited at 285 nM in the presence of 1 mM Ca2+ and either 25% PS/75% PC, 25% PS/75% 6–7-dibromo-PC, or 11–12-dibromo-PC. The number refers to the position of bromine quenchers on the sn-2 acyl chain of PC. F234W and M173W were quenched 36% and 29% by 11–12-dibromo-PC, and 52% and 56% by 6–7-dibromo-PC, respectively.
(B) Kinetics of C2A-F234W and C2A-M173W penetration into lipid bilayers. Measurements were made as described in Figure 4A using 5 μM protein, 100 μM Ca2+, and 10 nM vesicles. Insertion is sequential, Trp-234 inserts before Trp-173.
(C and D) Comparison of Trp insertion rates and FRET measurements of C2A-F234W and C2A-M173W. Trp insertion was monitored as described in (B) above. FRET was monitored by exciting the Trp residue at 285 nm and measuring the emission of a dansyl-phosphatidyl ethanolamine (PE) acceptor in the vesicles (composed of 25% PS/70% PC/5% dansyl-PE) via a 523 nm band-pass filter. Conditions were 5 μM protein, 100 mM Ca2+, and 10 nM vesicles. Trp insertion and binding measured via FRET are virtually simultaneous.
Neuron  , DOI: ( /S (00) )

3. Figure 1
Monitoring Ca2+ and Vesicle Binding to the C2A Domain of Synaptotagmin in Solution
(A) Structure of the C2A domain of synaptotagmin, in which three bound Ca2+ ions are shown (modified from Shao et al., 1998, using MOLSCRIPT [Kraulis 1991]). Two versions of C2A were generated, one with a Trp substitution at position 234, within Ca2+–binding loop 3, and another with a Trp placed in position 173, within Ca2+–binding loop 1.
(B) Spectral properties of Trp substitution mutants. Protein (10 μM) in HBS was excited at 285 nm in the presence of 1 mM EGTA. Ca2+ was then added (1 mM free) followed by addition of vesicles (10 nM) composed of 25% PS/75% PC. Fluorescence of the Trp-234 reporter is slightly decreased upon addition of Ca2+ (9%); fluorescence of the Trp-173 reporter is significantly increased by Ca2+ (27%). Both reporters show large increases in fluorescence (51% and 103% for Trp-234 and Trp-173, respectively, compared with the intensity in EGTA) and blue-shifts in their emission spectra upon binding vesicles. Vesicles did not influence the spectra in the absence of Ca2+.
(C) C2A–Ca2+–vesicle interactions are rapid and reversible. Samples were prepared and excited as in (B), and the emission intensity was monitored versus time at 340 nm. Additions were as in (B), and the Ca2+ effect was reversed by addition of 2 mM EGTA.
Neuron  , DOI: ( /S (00) )

4. Figure 2 Ca2+ Sensitivity of Wild-type and C2A Reporter Constructs
(A) C2A-Trp reporter mutations do not affect the Ca2+ dependence of vesicle binding. Wild-type (triangles), M173W (circles), and F234W (squares) C2A domains were immobilized as GST fusion proteins on glutathione-Sepharose beads at low-protein density (0.3 μg/μl) and assayed for binding to 3H-labeled liposomes (25% PS/75% PC) as a function of [Ca2+]free. Data were normalized, plotted, and fit with GraphPad Prism 2.0 software. In all cases, the [Ca2+]1/2 values (21, 21, 16 μM) and Hill coefficients (2.4, 1.8, and 2.8) were similar for wild-type, F234W, and M173W C2A domains, respectively.
(B) C2A-Trp reporter mutations do not affect the extent of vesicle binding. Wild-type (WT), M173W, or F234W mutant C2A domains (15 μg) were immobilized as GST fusion proteins and assayed for vesicle binding (1.75 μg total 3H-labeled lipids) in 2 mM EGTA (−) or 1 mM Ca2+ (+) in 150 μl HBS, as described (Chapman and Davis 1998).
(C) Ca2+ dependence of C2A-F234W-vesicle interactions measured in solution. The emission spectra shown in Figure 1B were corrected, integrated, and plotted as a function of [Ca2+]free ([Ca2+]1/2 = 74 μM Ca2+, Hill coefficient = 1.9). In all experiments, error bars represent the standard deviations from a minimum of three independent determinations.
Neuron  , DOI: ( /S (00) )

5. Figure 3
Steady-State and Kinetic Measurements of Ca2+ Binding to the C2A Domain of Synaptotagmin
(A) The Ca2+-induced change in C2A-M173W fluorescence was exploited to measure the dissociation constant for this binding site(s). C2A-M173W (5 μM) was excited at 285 nm, and spectra were collected from 300–400 nm as a function of [Ca2+]. Spectra were integrated, corrected, normalized, and plotted (closed circles) versus [Ca2+]free. Data were fit with GraphPad Prism 2.0 software. The Kd was 61 μM, consistent with NMR studies of Ca2+–binding site 1 (Shao et al. 1996); the Hill coefficient was 1.1. For comparison, the amplitudes of the kinetic traces in (B) are also plotted versus [Ca2+]free (open circles), demonstrating the agreement between the steady-state and kinetics experiments.
(B) Kinetics of Ca2+ binding to C2A-M173W. This series of stopped-flow experiments was carried out at 4°C using 5 μM protein. The sample was excited at 285 nm and emitted light collected with a 335 nm cutoff filter. Amplitudes of response were normalized and plotted versus time. At the lowest [Ca2+] tested, equilibrium was reached within the dead time of the instrument (1.2 ms), providing a lower limit for the second-order rate constant of ∼108 M−1s−1.
Neuron  , DOI: ( /S (00) )

6. Figure 4
Assembly and Disassembly Kinetics of C2A–Ca2+–Vesicle Complexes
The fluorescence change exhibited by C2A-F234W upon Ca2+-triggered binding to vesicles was used to monitor the kinetics of C2A–Ca2+–vesicle interactions. C2A-F234W was excited at 285 nm, and emitted light was collected with a nm band-pass filter. All kinetics data are shown referenced to an arbitrary time = 0. The 2 ms plateau phase at the beginning of each trace corresponds to the signal, which is collected during the “flow” and prior to the “stop”; the dead time was 1.2 ms.
(A) Binding of C2A-F234W-Ca2+ to vesicles in real time. Vesicles (25% PS/75% PC; 11 nM final concentration) were premixed with Ca2+ (100 μM final concentration) and then rapidly mixed with C2A-F234W (5 μM final concentration). As a control, mixing experiments were also carried out using 2 mM EGTA instead of Ca2+ (lower trace), providing a true minimum reference point. Data (1000 points) were collected for 100 ms and are plotted with a best-fit single exponential function (kobs = 312 s−1). The first 10 ms are shown on an expanded timescale in the inset.
(B) Determination of kon and koff for the C2A-F234W-vesicle complex in the presence of Ca2+. Stopped-flow experiments were carried out as in (A), and kobs was determined by fitting the data with single exponential functions, and plotted versus [vesicle]. The Y intercept yields koff for the C2A-F234W-vesicle complex in the presence of Ca2+ (240 s−1), and the slope yields kon (0.8 × 1010 M−1s−1) for binding of C2A-F234W-Ca2+ to vesicles (Equation 1). Error bars represent standard deviations from four independent experiments.
(C) Disassembly kinetics of the C2A-F234W-Ca2+-vesicle complex upon chelation of Ca2+. C2A-F234W-Ca2+-vesicle complexes were assembled at 100 μM Ca2+ with vesicles composed of 25% PS/75% PC. Disassembly reactions were carried out by rapidly mixing these complexes with 5 mM EGTA (final). As a control, samples were mixed with buffer lacking EGTA, thus providing a maximum signal reference. Disassembly data were collected for 100 ms; the plot includes a best-fit single exponential function. The first 10 ms are shown on an expanded timescale in the inset.
(D) Model for C2A–Ca2+–vesicle dynamics. Summary of the rate constants: kon-Ca2+ ≥ 108 M−1s−1; koff-Ca2+ ≥ 6 × 103 s−1 (determined from kon-Ca2+ and a Kd of 61 μM; Figure 3A; Shao et al. 1996); kon ≈ 1010 M−1s−1; koff = 240 s−1 (in the presence of 100 μM Ca2+ with vesicles composed of 25% PS/75% PC); kdiss = 700 s−1 (for complexes assembled at 100 μM Ca2+ with vesicles composed of 25% PS/75% PC).
Neuron  , DOI: ( /S (00) )

7. Figure 6
Comparison of the Kinetics of the C2A Domain of Synaptotagmin I with Other C2 Domains and Kinetics of C2B Domain–Mediated Oligomerization
(A and B) Association (A) and disassembly (B) kinetics of the C2A-F234W (derived from synaptotagmin I; syt I), synaptotagmin III (syt III; Mizuta et al. 1994; Fukuda et al. 1995), PKCβ (Shao et al. 1996), and cPLA2 (Nalefski et al. 1997; Perisic et al. 1998) were examined by monitoring Trp fluorescence as a function of time as described in Figure 4. Complexes were assembled with 1 mM [Ca2+] (final) and disassembled with 5 mM EGTA. Vesicles (11 nM) were composed of 25% PS/75% PC. Because of the differences in the kinetics of each C2 domain, data were collected over a logarithmic timescale.
(C) Kinetics of Ca2+-induced C2B-mediated synaptotagmin oligomerization. For these experiments, the cytoplasmic domain of recombinant oligomerization-competent synaptotagmin (8 μM) was rapidly mixed with 1 mM Ca2+ (upper trace) or 2 mM EGTA (lower trace) in HBS. Clustering of synaptotagmin was monitored by illuminating at 335 nm and collecting scattered light via a 335 nm cutoff filter. The light-scatter signal is shown on an expanded time frame in the inset. Mg2+ did not trigger oligomerization and served as a negative control (data not shown).
Neuron  , DOI: ( /S (00) )

8. Figure 7
Ca2+ Triggers Simultaneous Binding of Synaptotagmin to Membranes and to the Four Helix Core of the SNARE Complex
(A) The “mini” SNARE complex is SDS resistant. Mini SNARE complex (1.5 μg) was dissociated into its component parts (residues 1–96 of synaptobrevin, 1–80 and 120–206 of SNAP-25, and 180–262 of syntaxin) by boiling in SDS sample buffer.
(B) The ability of synaptotagmin (2.5 μM) to bind to the mini complex (3 μM) in 2 mM EGTA (−Ca2+) or 1 mM Ca2+ (+Ca2+) was assayed by coimmunoprecipitation with anti-synaptobrevin antibodies as described in the Experimental Procedures. Note: in all immunoprecipitation experiments, a nonoligomerizing version of synaptotagmin I was employed (see Experimental Procedures for details).
(C) Synaptotagmin–Ca2+–mini complexes were assembled (at 4 μM synaptotagmin and 4 μM mini complex) and purified by immunoprecipitation as described in (B). In parallel, controls lacking either SNARE complexes or synaptotagmin were also prepared. The immunoprecipitated complexes were then assayed for radiolabeled liposome-binding activity as described in Figure 2B. Error bars represent the standard deviation from triplicate determinations.
Neuron  , DOI: ( /S (00) )

9. Figure 8
The H3 Domain of Syntaxin Contains the Sole Synaptotagmin-Binding Site
(A) Binding of native synaptic proteins to syntaxin truncation and deletion mutants. GST–syntaxin fusion proteins (20 μg) were immobilized with glutathione-Sepharose and incubated with synaptosomal Triton X-100 extracts (1 mg protein at 1 mg/ml) in the presence of 2 mM EGTA (−) or 1 mM Ca2+ (+) for 2 hr at 4°C. Beads were washed and subjected to SDS–PAGE and immunoblot analysis to detect synaptotagmin and, as controls, SNAP-25, synaptobrevin II, α/β-SNAP, and synaptophysin. Immunoreactive bands were visualized with enhanced chemiluminescence. Twenty-five percent of the bound material and 6 μg extract (total) were analyzed.
(B) Binding of soluble syntaxin fragments to the immobilized C2A domain of synaptotagmins I and IV. Full-length (0.5 μM), 1–177 (10 μM), and 180–288 (0.5 μM) fragments of syntaxin were prepared as described in the Experimental Procedures and incubated with 20 μg GST, GST-C2A-I, or GST-C2A-IV in 150 μl TBS plus 0.5% Triton X-100 in either 2 mM EGTA (−Ca2+) or 1 mM Ca2+ (+Ca2+) for 2 hr at 4°C. Pellets (30%) were loaded onto the gel; “total” corresponds to 100 ng of full-length syntaxin and 50 ng of the 1–177 and 180–288 fragments.
(C) Schematic representation of the regions of syntaxin that interact with other proteins. Helical regions (designated by “H”) are shaded. Ha, Hb, and Hc correspond to helical domains that assemble into a three-stranded helical bundle (Fernandez et al. 1998), and H3 corresponds to a region that forms a helix within the ternary SNARE complex (Sutton et al. 1998). The closed rectangle corresponds to the transmembrane domain. Binding domains for other binding proteins are indicated with bars (Betz et al. 1997, and references therein; Fujita et al. 1998; Naren et al. 1998).
Neuron  , DOI: ( /S (00) )

10. Figure 9
Both C2 Domains of Synaptotagmin Are Required for High-Affinity Syntaxin-Binding Activity
(A) Binding of synaptotagmin C-terminal truncation mutants to syntaxin. Full-length His6-syntaxin (syx; 0.5 μM) was incubated with recombinant fragments of synaptotagmin (stg; 2.5 μM) in 170 μl TBS plus 0.5% Triton X-100 in the presence of 2 mM EGTA (-Ca2+) or 1 mM Ca2+ (+Ca2+). As a control for nonspecific precipitation of synaptotagmin, samples were prepared lacking syntaxin (−syx). The sequence of each synaptotagmin fragment is indicated on the left. Synaptotagmin binding was assayed by coimmunoprecipitation with syntaxin as described in the Experimental Procedures. Thirty percent of the immunoprecipitate (IP) and 0.5 μg of synaptotagmin were subjected to SDS–PAGE and stained with Coomassie blue. Abbreviation: L-chain, light chain from the anti-syntaxin monoclonal antibody.
(B) Ca2+ binding to C2A is essential for promoting synaptotagmin–syntaxin interactions. Aspartate residues 230 and 232 were neutralized by substitution with asparagines, and the ability of the mutant cytoplasmic domain of synaptotagmin to bind syntaxin analyzed by coimmunoprecipitation as described in (A). The D230,232N mutant failed to bind syntaxin.
(C) Schematic diagram indicating the primary sequence of synaptotagmin that is required to form high-affinity complexes with syntaxin. The closed rectangle corresponds to the transmembrane domain; the C2 domains are shaded. Residues 96–139 are not required for C2A–syntaxin interactions (Li et al. 1995; data not shown); therefore, the syntaxin-binding domain is shown extending from residues 140–337.
Neuron  , DOI: ( /S (00) )