Membrane-Binding Cooperativity and Coinsertion by C2AB Tandem Domains of Synaptotagmins 1 and 7

Abstract

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together sense Ca²⁺ to bind membranes and promote the stabilization of exocytotic fusion pores. Syt-1 triggers fast release of neurotransmitters, whereas Syt-7 functions in processes that involve lower Ca²⁺ concentrations such as hormone secretion. Syt-1 C2 domains are reported to bind membranes cooperatively, based on the observation that they penetrate farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously suggested that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we investigated C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. Equilibrium Ca²⁺ titrations demonstrate that the Syt-7 C2AB tandem binds liposomes lacking phosphatidylinositol-4,5-bisphosphate (PIP₂) with greater Ca²⁺ sensitivity than either of its individual domains and binds to membranes containing PIP₂ even in the absence of Ca²⁺. Stopped-flow kinetic measurements show differences in cooperativity between Syt-1 and Syt-7: Syt-1 C2AB dissociates from PIP₂-free liposomes much more slowly than either of its individual C2 domains, indicating cooperativity, whereas the major population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, suggesting a lack of cooperativity. A minor subpopulation of Syt-7 C2AB dissociates at a slower rate, which could be due to a small cooperative component and/or liposome clustering. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts deeply into membranes as part of the C2AB tandem, similar to the coinsertion previously reported for Syt-1. Overall, coinsertion of C2A and C2B domains is coupled to cooperative energetic effects in Syt-1 to a much greater extent than in Syt-7. The difference can be understood in terms of the relative contributions of C2A and C2B domains toward membrane binding in the two proteins.

Figure 1

Schematic structure of synaptotagmin. (A) Full-length synaptotagmins contain an N-terminal transmembrane helix anchored to either the secretory vesicle or plasma membrane (65, 66) and two C-terminal C2 domains. (B) The Syt-1 and Syt-7 C2AB protein fragments used in this study consist of the C-terminal C2 domains, which coordinate Ca²⁺ ions and insert into membranes primarily via Ca²⁺-binding loops (CBL1 and CBL3). The C2B domains additionally contain a polybasic patch (blue shading) that can interact with anionic lipids in a Ca²⁺-independent manner. To see this figure in color, go online.

Figure 2

Ca²⁺ sensitivity of Syt-7 (A and B) and Syt-1 (C) C2 domains. CaCl₂ was titrated into solutions containing the indicated C2A (triangles), C2B (squares), or C2AB (circles) domains and liposomes. Data were normalized based on best fits to Eq. 1; the parameters from these fits are given in Table 2. Points and error bars shown are mean ± SD of three replicate titrations; where not visible, error bars are smaller than the symbol. Right panels show the extent of reversibility upon addition of 2.5 mM EDTA.

Figure 3

Dissociation kinetics of Syt-1 C2 domains on (A) 1:1 DOPC/DOPS liposomes and (B) PM(−)PIP₂ liposomes. Dansyl-PE fluorescence was monitored as solutions containing 5 μM (single domains) or 1 μM (tandem domain) protein, 200 μM CaCl₂, and liposomes (200 μM accessible lipid) were rapidly mixed with an equal volume of 2 mM EDTA (all concentrations listed are before mixing). Kinetic data were fitted to Eqs. 2 or 3, with rate constants shown in Table 3. Inset shows the full timescale of measurement of Syt-1 C2AB. Data shown are representative of ≥3 independent measurements.

Figure 4

Dissociation kinetics of Syt-7 C2 domains on (A) 1:1 DOPC/DOPS liposomes and (B) PM(−)PIP₂ liposomes. Dansyl-PE fluorescence was monitored as solutions containing 1 μM protein (in A) or 0.25 μM protein (in B), 200 μM CaCl₂, and liposomes (200 μM accessible lipid) were rapidly mixed with an equal volume of 2 mM EDTA (all concentrations listed are before mixing). Kinetic data were best fitted to Eqs. 2 or 3, with rate constants listed in Table 3. Data shown are representative of ≥3 independent measurements.

Figure 5

Membrane insertion of Syt-7 C2A and C2B domain loops in the absence of PIP₂. Fluorescence emission spectra are shown of Syt-7 single domains (A, C, E, and G) or the C2AB tandem (B, D, F, and H) labeled with AEDANS on CBL1 (A, B, E, and F) or CBL3 (C, D, G, and H) (see Fig. S9 for schematic of labeling sites and Materials and Methods for residue numbers). Data are of protein in solution alone (black) and after addition of PM(−)PIP₂ liposomes (gray). All spectra are normalized to the maximal intensity in the absence of lipid.

Figure 6

Structural model of linkage effects on C2AB insertion and dissociation kinetics. (Left) The individual C2A domains of Syt-1 (top) and Syt-7 (bottom) penetrate membranes primarily via their CBLs (20, 21, 22, 23, 24) (Fig. 5, A and C); Syt-1 C2A dissociates much faster than Syt-7 C2A upon addition of EDTA (16) (Table 3). (Center) The individual C2B domains bind membranes relatively superficially through interaction of the polybasic region (blue shading) with lipid headgroups (30) along with modest insertion of CBL3 (23) (Fig. 5, E and G). (Right) In the C2AB tandem, linkage to C2A affects the binding geometry of the C2B domain, leading to deeper insertion of its CBL1 and CBL3 (23, 24) (Fig. 5, F–H). For Syt-1, this deeper insertion leads to much slower dissociation kinetics compared to the individual domains, i.e., interdomain cooperativity. For Syt-7, dissociation kinetics are dominated by the C2A domain and relatively unaffected by C2A-C2B linkage (Fig. 4). (Bottom right) A minor, very slow phase in Syt-7 C2AB dissociation kinetics may be explained by liposome clustering, which could include a subpopulation of C2AB fragments bound in trans to opposing liposomes. Brown circles represent Ca²⁺. Curvature is approximately to scale of 100-nm-diameter liposomes (Fig. S7). Kinetics upon EDTA addition are as follows: very fast, >500 s⁻¹; fast, 80–200 s⁻¹; moderate, 10–50 s⁻¹; slow, 1–10 s⁻¹; very slow, <1 s⁻¹ (Table 3). To see this figure in color, go online.