Schematic summary of C2AB interactions with SNAP25. The presence of Ca²⁺ altered interactions between C2A and C2B domains with SNAP25. For C2A, residues 191-196 bound the C terminal portion of the N terminal helix of SNAP25 (residues 77-83) in the absence but not presence of Ca²⁺ (orange). In the presence of Ca²⁺, C2A residues 191–200 bound the C terminal helix of SNAP25 (residues 176-201; blue). For C2B, residues 301-313 bound to the N terminus of SNAP25 (residues 32-40; green) in the absence of Ca²⁺ but shifted to C2B residues 289-297 in the presence of Ca²⁺.

Membrane-docked model for Ca²⁺-dependent C2A-SNARE interaction. (a) Space-filling model of C2A depicting clustered basic residues of β4-loop 2 (K196, H198, R199, and K200) positioned orthogonally to membrane-inserting (M173 and F234) residues of loops 1 and 3. R233 is shown as positioned between both interfaces. (b) Model depicts simultaneous interactions of C2A with SNARE complex and membrane. Ca²⁺ ions (green spheres) bound by loops 1 and 3 with F234 penetrating the bilayer are shown. The orthogonal face containing K196, R199, and K200 interacting with SNAP25 C-terminal helix is depicted. R233 is shown between membrane- and SNARE-binding interfaces.

C2A loop 2 and C2B loop 1 mutants exhibit decreased Ca²⁺-dependent SNARE binding but normal Ca²⁺-dependent phosphatidylserine binding. (a) C2AB, C2AB(R199A/K200A) (RK) or C2AB(R199A/K200A/K297A/K301A) (RK/KK) were incubated with protein-free PC liposomes containing 15% PS at the indicated Ca²⁺ concentrations and bound C2AB was determined as in Figure 3b. Maximal bound C2AB was set equal to 100% and plotted (±SE, n = 3) as a function of [Ca²⁺]. (b) C2AB, C2AB(R199A/K200A) (RK), or C2AB(R199A/K200A/K297A/K301A) (RK/KK) were incubated with protein-free PC liposomes containing 5% PIP₂ at the indicated Ca²⁺ concentrations and bound C2AB was determined as in Figure 3b. Fractional bound C2AB was plotted (±SE, n = 3) as a function of [Ca²⁺]. (c) C2AB and indicated mutants were incubated with PC liposomes containing SNARE complexes at the indicated Ca²⁺ concentrations, and bound C2AB was determined as in Figure 3b. The fraction of C2AB bound was plotted (±SE, n = 3) as a function of [Ca²⁺].

Ca²⁺ stimulates the formation of a C2AB-SNAP25 cross-linked adduct. (a) Wild-type C2AB (not shown) or C2AB K326A/K327A (1 μM) and SNAP25 (1 μM) were incubated in the absence (−) or presence (+) of 1 mM CaCl₂ before the addition of cross-linker (EDC/sulfo-NHS) and analyzed by SDS PAGE with Coomassie staining. C2AB K326A/K327A was used to minimize oligomerization (Chapman et al., 1998), but identical results were obtained with wild-type C2AB and no self cross-linking of C2AB was observed. Bands corresponding to SNAP25, C2AB, SNAP25 dimer, C2AB-SNAP25 adduct (*), and hsp70 are indicated. Lane on right was loaded with 10% input lacking cross-linker. (b) Both C2 domains are required for optimal cross-linking of C2AB to SNAP25. C2AB, C2A, or C2B fusion proteins (1 μM) were incubated with SNAP25 (1 μM) in the absence (−) or presence (+) of 1 mM Ca²⁺ before cross-linking. Bands corresponding to C2AB-SNAP25 (*), C2B-SNAP25, C2A-SNAP25, or free proteins are indicated.

Ca²⁺-dependent C2A/SNARE binding interface derived from computational modeling. (a) Using ZDOCK, each of 20 NMR structures of C2A was computationally docked to the atomic coordinates of the SNARE complex crystal structure. The frequency of occurrence of individual C2A residues within 5 Å of acidic C-terminal SNAP25 residues D179, D186, and D193 is depicted. C2A is shown in a space filling model docked to a ribbon model of the SNARE complex. C2A residues are colored according to their frequency of proximity to SNAP25 D179, D186, or D193 from 0 (blue) to 20 (red) of 59. (b) The model shows the predicted low-energy docking orientation for Ca²⁺-dependent C2A-SNARE binding. The model corresponds to complex 1–1826 shown in Supplementary Tables S1 and S2. Close-up view depicts the binding interface for synaptotagmin−1 residues (K196, R199, and K200) with SNAP 25 residues (D179, D186, and D193).

Synaptotagmin−1 mutants fail to restore Ca²⁺-dependent exocytosis in synaptotagmin-depleted PC12 cells. (a) Immunoblot analysis of wild-type PC12 cells, synaptotagmin−1/9-null cells, and null cells expressing wild-type synaptotagmin−1 or R199A/K200A (RK) or R199A/K200A/K297A/K301A (RK/KK) mutants. SNAP25 was used as a loading control. (b) Wild-type and synaptotagmin−1/9-null cells expressing synaptotagmin−1 or indicated mutants were fixed and stained with synaptotagmin−1 (green channel) and CgB (red channel) antibodies, and colocalization was determined (yellow). The synaptotagmin−1 mutants were localized to dense-core vesicles. (c) Cells expressing ANF-EGFP were stimulated with depolarization medium, and images were acquired at 0.25-s intervals. Fusion events were counted as a flash/puff of fluorescence for each cell type, and the sum of fusion events in 10-s intervals was determined. The sums of the average number of fusion events per cell over time (mean value ± SD) for wild-type (n = 25), synaptotagmin−1/9-null (n = 25), synaptotagmin−1/9-null + synaptotagmin−1 (n = 20), synaptotagmin−1/9-null + synaptotagmin−1 RK (n = 15), and synaptotagmin−1/9-null + synaptotagmin−1 RK/KK (n = 15) cells were plotted.

C2A loop 2 and C2B loop 1 mutations reduce Ca²⁺ stimulation of liposome fusion mediated by C2AB. (a) C2AB, 10 μM, stimulated the fusion of v-SNARE with t-SNARE liposomes in the presence of 100 μM Ca²⁺ but not in the presence of 0.2 mM EGTA. NBD fluorescence was expressed as percentage maximum fluorescence after 1% dodecyl maltoside addition. (b) C2AB, C2AB(K297A), or C2AB(K297A/K301A), all at 10 μM, were tested in the liposome fusion assay. C2AB(K297A/K301A) exhibited partial loss of function in conferring Ca²⁺ stimulation. (c) C2AB(R199A/K200A) (RK) or C2AB (R199A/K200A/K297A/K301A) (RK/KK) mutants failed to stimulate liposome fusion in the presence of 100 μM Ca²⁺. (d) C2AB, 10 μM, and mutants were incubated in the liposome fusion assay with indicated [Ca²⁺]. % maximal NBD fluorescence at 120 min was plotted as a function of [Ca²⁺] (±SE, n = 3). EC₅₀ values for Ca²⁺ were 113 μM (±4 μM) for C2AB, 181 μM (±9 μM) for C2AB(R199A/K200A) (RK), and 187 μM (±9 μM) for C2AB (R199A/K200A/K297A/K301A) (RK/KK).

C2A loop 2 and C2B loop 1 mutations reduce Ca²⁺-stimulated binding of C2AB to SNARE complexes. (a) Wild-type C2AB, C2AB(R199A/K200A), C2AB (E194A/K196A) or C2AB(K191A/K192A), all at 1 μM, were incubated with 1 μM SNARE complex immobilized on glutathione-Sepharose beads in the absence (−) or presence (+) of 1 mM CaCl₂. Binding of C2AB was determined by Western blotting after SDS PAGE. Mean values ± SE are shown (n = 3). (b) C2AB, C2AB(R199A/K200A) (RK) or C2AB (R199A/K200A/K297A/K301A) (RK/KK), all at 10 μM, were incubated with SNAP25/syntaxin-containing PC liposomes in the absence or presence of 1 mM Ca²⁺. Liposome-bound C2AB was isolated from Accudenz gradients and analyzed by SDS-PAGE and Coomassie Blue staining. (c) C2AB and indicated mutants were incubated with SNAP25/syntaxin-containing liposomes in the presence or absence of 1 mM Ca²⁺. RK corresponds to R199A/K200A. Bound C2AB was analyzed as in panel b, quantified by densitometry, and normalized to syntaxin in each gradient. Ca²⁺-dependent binding by C2AB mutants was expressed as the difference bound in the presence or absence of Ca²⁺ and normalized to binding by wild-type C2AB. Mean values ± SE are shown (n = 3). Differences in binding between wild-type C2AB and RK or K297A/K301A mutants, or between RK and RK/K297A/K301A mutants were significant (**p < 0.01; *p < 0.05).