Direct Interaction of a Ca2+-binding Loop of Synaptotagmin with Lipid Bilayers*

Edwin R. ChapmanDagger and Anson F. Davis

From the Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Synaptotagmin 1 binds Ca2+ and membranes via its C2A-domain and plays an essential role in excitation-secretion coupling. In this study, we sought to identify Ca2+- and membrane-induced local conformational changes in the C2A-domain of synaptotagmin and to delineate the C2A-lipid binding interface. To address these questions native phenylalanine residues were replaced, at each face of the domain, with tryptophan reporters. Changes in tryptophanyl fluorescence indicated that Ca2+ induced long range conformational changes throughout C2A, including regions distant from an established Ca2+-binding site. Addition of liposomes resulted in Ca2+-dependent increases in the fluorescence of tryptophans 193, 231, and 234. Only the tryptophan residues at positions 234 and 231, which lie within a Ca2+-binding loop of C2A, exhibited liposome-induced blue shifts in their emission spectra. Quenching experiments, using membrane-imbedded doxyl spin labels, revealed that tryptophan residues 231 and 234 penetrated lipid bilayers. These data delineate the lipid binding interface of C2A and provide the first evidence for adjacent Ca2+- and lipid-binding sites within a C2-domain. The penetration of C2A into membranes may function to bring components of the fusion machinery into contact with the lipid bilayer to initiate exocytosis.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Neuronal exocytosis is strictly regulated by Ca2+ ions (1). Ca2+ triggers the fusion of synaptic vesicles with the presynaptic plasma membrane on the submillisecond time scale, indicating that a limited number of conformational changes couple Ca2+ to exocytotic membrane fusion (2, 3). While a number of proteins that function in exocytosis have been identified (4-6), little is known concerning the Ca2+-driven conformational changes or protein-lipid interactions that catalyze lipid bilayer fusion. The key to unraveling this mechanism lies in the Ca2+ sensor for exocytosis. Recent gene disruption experiments (7-10) have established that the Ca2+-binding synaptic vesicle protein, synaptotagmin 1 (11-13), is essential for rapid and efficient Ca2+-triggered release of neurotransmitters. Thus, synaptotagmin is has been proposed to function as the major Ca2+ sensor of regulated exocytosis.

Synaptotagmin is represented by a growing gene family, which currently contains 11 members (14-16). The members of this family span the vesicle membrane once and have a short carboxyl-terminal intravesicular domain and a large cytoplasmic region that contains two C2-domains, designated C2A and C2B. C2A is the membrane proximal domain and mediates the interaction of synaptotagmin with anionic phospholipids (17, 18). This interaction has been proposed to contribute to the lipid rearrangements that underlie membrane fusion (13). The membrane distal C2B-domain mediates Ca2+-dependent oligomerization of synaptotagmin, potentially clustering the release machinery into a collar or ring-like structure (19-21). Finally, both C2-domains are required for high affinity binding to syntaxin (19). The synaptotagmin-syntaxin interaction is regulated by Ca2+ concentrations similar to those required for neuronal exocytosis (22). In addition, syntaxin is an essential component of the putative membrane fusion machinery (23-25). Thus, the synaptotagmin-syntaxin interaction may function at the triggering step in exocytosis.

The C2-domains of synaptotagmin mediate a variety of Ca2+-dependent effector interactions. More generally, C2-domains are found in more than 50 distinct proteins, including lipid and serine/threonine kinases, phospholipases, GTPase-activating proteins, and a number of proteins involved in membrane traffic (26). Despite the growing number of proteins that possess C2-domains, relatively little is known concerning the structural elements that mediate C2-domain-effector interactions. A number of C2-domains bind membranes in a Ca2+-dependent manner (17, 18, 27-34). Because the interaction of the C2A-domain of synaptotagmin with membranes is likely to serve as an important step in excitation-secretion coupling, we have probed for conformational changes in synaptotagmin, induced by Ca2+ and lipids, and have sought the region of C2A that engages lipid bilayers. To address these issues we employed fluorescence spectroscopy to probe the environment of tryptophan (Trp) "sensors" placed in different locations within C2A. The sensitivity of indole side chain fluorescence to local environment was exploited to monitor local conformational changes as a consequence of binding Ca2+ and lipids. Using membrane-imbedded nitroxide quenchers, we provide direct evidence for the partial insertion of a Ca2+-binding "jaw" of C2A into the lipid bilayer. These findings are discussed in the context of the mechanism of regulated membrane fusion.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Site-directed Mutagenesis and Preparation of Recombinant Proteins-- cDNA encoding rat synaptotagmin 1 was kindly provided by T. C. Südhof (Dallas, TX) (12). C2A-W259 encodes residues 96-265 and contains a single naturally occurring Trp residue at position 259. All other constructs encode residues 96-258 and therefore lack native Trp residues. Phe-234, Phe-231, Phe-193, and Phe-153 were individually changed to Trp residues using site-directed mutagenesis as described previously (18). All C2A-domains were subcloned into pGEX-2T and expressed as glutathione S-transferase (GST)1 proteins using glutathione-Sepharose (Amersham Pharmacia Biotech) as described (35). Soluble domains, for fluorescence studies, were generated by thrombin cleavage of fusion protein coated beads (19). Proteins were quantified by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue using bovine serum albumin as a standard.

Liposome Binding Assays-- Brain-derived phosphatidylserine (PS) and phosphatidylcholine (PC) and synthetic 1-palmitoyl-2-stearoyl (n-doxyl)-sn-glycero-3-phosphocholine (doxyl-PC), with the spin labels at the 5-, 7-, or 12-position of the sn-2 acyl chain, were obtained from Avanti Polar Lipids. L-3-phosphatidyl[N-methyl-3H]choline-1,2-dipalmitoyl ([3H]PC) was purchased from Amersham Pharmacia Biotech. [3H]Phospholipid binding assays were carried out as described (17, 36). For fluorescence studies, large (~100 nm) unilamellar liposomes (37) were prepared using an Avanti Polar Lipids extruder according to the manufacturer's instructions. For all experiments, the actual lipid concentration was determined by measuring total phosphate as described (38).

Fluorescence Measurements-- Fluorescence measurements were made at 24 °C using a PTI QM-1 fluorometer and Felix software. Protein samples were adjusted to 5 µM in Tris-saline (TS; 20 mM Tris, pH 7.4, 150 mM NaCl) and excited at 288 nm with 2-nm resolution. Emission spectra were collected from 300 to 450 nm and were corrected for blank and dilution due to the addition of EGTA, Ca2+, or liposomes.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Tryptophan fluorescence is strongly influenced by the environment of the indole side chain and has thus proved to be a useful tool to study conformational changes in proteins, protein-protein interactions, and protein-membrane interactions. For example, in hydrophobic environments, Trp fluorescence generally shifts to shorter wavelengths and often exhibits increases in intensity. The goal of the present study was to place Trp reporters at different surfaces of C2A to assay for local Ca2+- and lipid-induced conformational changes and to delineate the C2A-lipid binding interface. We relied on the crystal structure of C2A reported by Sutton et al. (39) to place Trp reporters in distinct regions of C2A (Figs. 2 and 3). Phe to Trp substitution mutations, as opposed to labeling engineered cysteine residues with exogenous fluorescent tags, was carried out in order to minimize the effects of the reporter group on the structure of C2A. This approach was facilitated by the paucity of Trp residues in C2A; the only naturally occurring Trp residue is at position 259. The fluorescence of this residue is included in the analysis described below. In addition, a series of recombinant C2A-domains were prepared that lacked residue 259 and contained either no Trp residues or single Phe to Trp substitution mutations at position 234, 231, 193, or 153. It should be noted that the C2-domain of protein kinase C-beta , which exhibits Ca2+-dependent lipid binding properties identical to C2A (31), has a Trp residue in a position analogous to residue 231 in C2A (26). Therefore, we can expect that this mutation will be tolerated.

To determine whether the Phe to Trp mutations affected Ca2+-dependent phospholipid binding activity, wild type and mutant recombinant C2A-domains were expressed as GST fusion proteins and immobilized using glutathione-Sepharose. The ability of immobilized C2A-domains to bind radiolabeled liposomes was analyzed as described (17, 36). As shown in Fig. 1, Ca2+ promoted the binding of similar levels of radiolabeled liposomes to each of the immobilized GST-C2A fusion proteins, confirming that the activity of the Trp substitution mutants remained intact.


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Fig. 1.   Trp substitution mutants of the C2A-domain of synaptotagmin 1 retain Ca2+-dependent liposome binding activity. Six versions of the C2A-domain of synaptotagmin were prepared: wild type C2A (Trp-259) contains a sole Trp residue at position 259, all other constructs lack this residue and contain either no Trp substitution mutations (-W) or Phe to Trp substitution mutations at positions 234 (F234W), 231 (F231W), 193 (F193W), or 153 (F153W). Recombinant wild type or mutant C2A-domains were prepared as GST fusion proteins as described under "Experimental Procedures." A, Coomassie-stained SDS-PAGE gel containing 2 µg of each recombinant fusion protein. B, 3H-labeled liposome binding assays were carried out as described (17, 36) using 15 µg of each recombinant protein and [3H]PC-labeled liposomes composed of 25% PS, 75% PC. Assays were carried out in triplicate; error bars indicate the S.D. Note that equivalent levels of labeled liposomes bound to wild type and mutant C2A-domains.

For fluorescence studies, the C2A-domains were cleaved from GST using thrombin. Soluble C2A-domains were adjusted to 5 µM, and the Trp residues were excited at 288 nm. Emission spectra were monitored from 300 to 450 nm in the presence of 2 mM EGTA, 1 mM free Ca2+, or 1 mM free Ca2+ plus liposomes composed of PS/PC. As shown in Fig. 2, a C2A-domain without a Trp residue exhibited low levels of short wavelength residual fluorescence resulting from the emission of Tyr and Phe side chains. Greater than 90% of the fluorescence of each Trp-containing C2A-domain was due to the emission of the indicated Trp residue. In EGTA, the emission intensity of Trp residues in different positions of C2A varied by more than a factor of three, while the emission maximum of each Trp was essentially the same (339-340 nm). Addition of Ca2+ affected the emission intensity of each Trp reporter with the exception of Trp-259. Trp residues 234, 231, and 153 exhibited slight quenching, while the emission of Trp-193 was increased by Ca2+. In addition, Ca2+ induced slight changes in the emission maximum of each of the Trp reporter groups (Table I). It is notable that Trp-153 lies at the opposite end of C2A relative to the Ca2+-binding jaws (Fig. 2). These findings suggest that Ca2+ induces conformational changes that are transmitted throughout the C2A-domain of synaptotagmin. It cannot be ruled out, however, that the effect of Ca2+ on the fluorescence of Trp-153 may be influenced by additional low affinity Ca2+-binding sites within C2A.


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Fig. 2.   Trp fluorescence reports Ca2+- and liposome-induced changes in the conformation and local environment of distinct regions of the C2A-domain of synaptotagmin. Soluble C2A-domains were prepared by thrombin cleavage of the immobilized fusion proteins shown in Fig. 1A. Proteins were adjusted to 5 µM in Tris-saline, pH 7.4, and aromatic residues were excited at 288 nm. The Trp emission spectra were collected from 300 to 450 nm using a PTI QM-1 fluorometer. Spectra were first obtained in the presence of 2 mM EGTA, followed by the addition of Ca2+ to a free concentration of 1 mM and finally after subsequent addition of liposomes composed of 50% PS, 50% PC (250 µM total lipid). Fluorescence intensity is given in arbitrary units. Note: liposomes had no effect on the Trp emission spectra in the absence of Ca2+ (data not shown). Furthermore, this effect was specific for Ca2+ and was not mimicked by Mg2+. These findings indicate that C2A first binds Ca2+ followed by association with membranes. Right panel, the crystal structure of C2A was rendered using MolScript (52) and Raster3D (53) according to a description file modified from Sutton et al. (39). Trp-259, and the Phe residues (234, 231, 193, 153) that were changed to Trp reporters, are shown in black. Loops 1, 2, and 3 are indicated with arrows. Note that loops 1 and 3 form the Ca2+-binding jaws of C2A (39).

                              
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Table I
Effects of Ca2+ and liposomes on the tryptophanyl emission spectra of C2A-Trp substitution mutants
The emission maximum of each of the spectra shown in Fig. 2 are listed in the left panels of the table. The relative changes in fluorescence intensity from the integrated spectra are listed in the right panels of the table. These values are normalized to the integral of the spectra obtained in EGTA. PL, liposomes composed of 50% PS/50% PC.

Interestingly, in the presence of Ca2+, liposomes had significant effects on the emission spectra of Trp-193, Trp-231, and Trp-234 (Fig. 2). Liposomes did not affect the fluorescence of Trp-259 or Trp-153 (Fig. 2). Most notably, residues 234 and 231 exhibited increases in fluorescence intensity and also exhibited blue-shifted emission maxima. The Ca2+-induced increase in the emission of Trp-193 was also enhanced by artificial membranes. However, no shift in the emission maximum of Trp-193 was observed. These findings indicate that the region surrounding Trp-193 undergoes a considerable conformational change due to the binding of Ca2+. This finding, coupled to the lower affinity of synaptotagmin for Ca2+ in the absence of lipids (13), suggests that the lipid effects may be due to an enhancement of Ca2+ binding to F193W. The effects of Ca2+ and liposomes on the emission maximum and fluorescence intensity of each Trp reporter are tabulated in Table I.

The liposome-induced blue shifts and increases in fluorescence intensity of Trp-234 and Trp-231 suggest that upon formation of the C2A-Ca2+-lipid complex, these residues shifted to a more hydrophobic environment. This relatively hydrophobic environment could lie either within the protein or within the lipid bilayer. To discriminate between these possibilities, we examined the ability of lipid embedded nitroxide spin labels to quench the fluorescence of each of the Trp reporter groups. Only Trp residues that can interact directly with the lipid bilayer will make contact with the nitroxide spin groups and become quenched. For these experiments, liposomes were prepared that contained doxyl-PC labeled at either the 5-, 7-, or 12-positions of the sn-2 acyl chain. These lipid-embedded quenchers, and their distances from the center of the lipid bilayer, are illustrated in Fig. 4. Recombinant C2A-domains were mixed with constant amounts of either control liposomes or liposomes containing 10% doxyl-PC, in the presence of Ca2+. The spectra from these experiments are shown in Fig. 3, and the degree of quenching by the nitroxide groups are tabulated in Table II. From these spectra, it is apparent that the doxyl spin labels efficiently quenched Trp residues placed in positions 231 and 234 and failed to quench Trp-153 or Trp-259. In addition, the fluorescence of Trp-193, which was enhanced by liposomes in the presence of Ca2+, was not quenched. We conclude that the changes in the local environment of Trp-193 are associated with interactions within the protein structure of C2A and not with the lipid bilayer. This finding supports the interpretation that the increase in fluorescence induced by liposomes is due to increased binding of Ca2+ to the F193W-C2A-domain.


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Fig. 3.   Trp residues within a Ca2+-binding jaw of C2A insert into the lipid bilayer in the presence of Ca2+. Liposomes composed of 50% PS, 50% PC or 50% PS, 40% PC, 10% doxyl-PC labeled at either the 5-, 7-, or 12-position of the sn-2 acyl chain were prepared and mixed with wild type and mutant C2A-domains (5 µM) in Tris-saline in the presence of 1 mM Ca2+. Spectra were obtained as described in Fig. 2. Note: doxyl-labeled liposomes had no effect on the Trp fluorescence of any of the reporters in the absence of Ca2+ (data not shown). Right panel, the structure of C2A was rendered as described in the legend to Fig. 2.

                              
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Table II
Quenching of tryptophanyl fluorescence of C2A-Trp substitution mutants by membrane-imbedded doxyl spin labels
All spectra from Fig. 3 were integrated and normalized relative to the fluorescence obtained using liposomes lacking the doxyl spin label. The percent quenching, relative to the total integrated fluorescence in the absence of the nitroxide spin labels, is listed.

The most striking finding was the efficient nitroxide quenching of both Trp reporters, Trp-234 and Trp-231, that are localized to Ca2+-binding loop 3 (31) of C2A (Fig. 3). These findings clearly establish a direct association with, and partial penetration into, lipid bilayers by this region of C2A. These data are summarized in the model shown in Fig. 4. Trp-234 penetrates more deeply into membranes, reaching doxyl groups that lie only 0.6 nm from the center of the bilayer. By contrast, Trp-231 is more readily quenched by doxyl labels that lie 1-1.2 nm from the bilayer center (Fig. 4). It should be noted that these are qualitative penetration measurements, with a high degree of uncertainty regarding the precise depth of penetration of loop 3 into membranes. However, these findings are consistent with the location of these residues; Trp-234 lies at the outermost edge of the Ca2+-binding loop, while Trp-231 lies at the base of the loop, close to the body of the C2A-domain. In contrast to our data, lipid-embedded bromine quenchers failed to quench the Trp fluorescence of intact protein kinase C-beta II (40). Because protein kinase C-beta II contains nine Trp residues, quenching of a small number of indole side chains may be difficult to detect. This problem was circumvented by the single Trp substitution mutants prepared and analyzed in the present study. Future studies on isolated C2-domains, derived from different parent proteins, will reveal whether these motifs interact with membranes in a manner consistent with their conserved primary sequence.


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Fig. 4.   Diagrammatic representation of the interaction of C2A with membranes. Doxyl spin labels are indicated at the 5-, 7-, and 12-positions of the sn-2 acyl chain of PC. The relative positions of the nitroxide quenchers from the center of the lipid bilayer are also indicated (54). Trp residues 231 and 234 are indicated at their relative depths in the membrane using brackets. At steady state, Trp-234 has equal access to the 5-, 7-, and 12 -positions, while Trp-231 is less accessible to all doxyl quenchers and is least quenched by doxyl groups at the 12-position. Thus, Trp-234 is more deeply imbedded in the lipid bilayer than Trp-231, which localizes more closely to the bilayer surface.

Crystallography studies indicate that portions of the C2 jaws of synaptotagmin and phospholipase C are highly mobile in both the free and Ca2+-bound states (39, 41). This mobility may account for the similar degrees of fluorescence quenching of Trp-234 by doxyl labels bound at different depths of the lipid bilayer. In addition, the steady state measurements shown in Fig. 3 are likely to reflect populations of C2A that penetrate the bilayer to varying extents. In any case, it is striking that of the five Trp substitution mutants characterized in the present study, only the Trp residues that lie within a Ca2+-binding loop of C2A exhibited liposome-induced blue shifts in their emission spectra and were quenched by membrane-imbedded nitroxide groups. Taken together, these findings demonstrate that this loop directly interacts with, and becomes partially inserted into, the lipid bilayer.

The insertion of Trp-234, and presumably the native Phe-234, into membranes suggested that C2A-membrane interactions may be mediated by hydrophobic forces. However, loop 3 contains numerous polar and charged side chains. In addition, structural studies prompted the proposal that the Ca2+-dependent interaction of C2-domains with effectors may be primarily mediated by electrostatic interactions (31, 41, 42). To further characterize the interaction of synaptotagmin with membranes, we analyzed the sensitivity of Ca2+-dependent synaptotagmin-lipid binding to ionic strength. For these studies we examined the interaction of the cytoplasmic domain of native synaptotagmin with synaptic vesicle membranes, as well as the interaction of C2A-F193W with artificial membranes, as a function of increasing [NaCl]. As shown in Fig. 5, the interaction of both native synaptotagmin and recombinant C2A with native and artificial membranes, respectively, was highly sensitive to ionic strength. Three-hundred mM NaCl was sufficient to inhibit greater than 75% of the binding. These data indicate that synaptotagmin-membrane interactions possess a high degree of ionic character. The molecular basis for these findings are discussed in greater detail below.


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Fig. 5.   Synaptotagmin-membrane interactions are sensitive to ionic strength. A: upper panel, the cytoplasmic domain of native synaptotagmin was generated by limited proteolysis and assayed for binding to proteolysed synaptic vesicle membranes by co-sedimentation as described (18) in the presence of 2 mM EGTA (-Ca2+) or 1 mM Ca2+ (+Ca2+). Immunoblots were carried out using the anti-synaptotagmin monoclonal antibody 41.1 and 125I-protein A as described (18). Addition of Ca2+ results in the complete translocation of the cytoplasmic domain of synaptotagmin from the supernatant (s) to the pellet (p) fraction. A: lower panel, co-sedimentation assays were repeated as in the upper panel in the presence of 1 mM Ca2+ and the indicated [NaCl]. Ca2+-dependent binding of the cytoplasmic domain of synaptotagmin to synaptic vesicle membranes was inhibited by 300 mM NaCl. B, the binding of C2A-F193W to membranes was determined by monitoring changes in the lipid induced increase in Trp-193 fluorescence in the presence of Ca2+ as a function of ionic strength. Spectra were integrated and the data normalized to the fluorescence observed at 150 mM NaCl. Seventy-five percent of the binding was inhibited by raising the [NaCl] to 300 mM. Similar results were obtained using F234W (data not shown).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Genetic studies have provided compelling evidence that synaptotagmin 1 functions as the major Ca2+ sensor in neuronal exocytosis (7-10). The ability of the C2A-domain of synaptotagmin to bind Ca2+ ions and thus trigger exocytosis is strongly potentiated by anionic phospholipids (13). The interaction of C2A with membranes may directly participate in the Ca2+-triggered lipid rearrangements that result in membrane fusion. However, this role would also involve interactions with components of the exocytotic fusion complex (discussed in detail below), because synaptotagmin itself does not function as a fusogen (13), and spontaneous membrane fusion events do not require synaptotagmin (8-10).

While little is known concerning the structure of C2-domain-Ca2+-effector complexes, the crystal structures of two C2-domain-Ca2+ complexes, the C2A-domain of synaptotagmin (39) and the C2-domain of phospholipase C-delta 1 (43), have been determined. These domains formed beta -sandwich structures with three loops that protrude from one end of the domain (indicated by arrows in Figs. 2 and 3). Initially, only a single Ca2+-binding site was identified in the C2A-domain of synaptotagmin (39). This site is formed by Ca2+ ligands that lie within loops 1 (Asp-172 and Asp-178) and 3 (Asp-230, Asp-232, and Asp-238). However, C2A crystals were destroyed by soaking in Ca2+ > 0.1 mM, presumably due to significant conformational changes, induced by the binding of additional Ca2+ ions, which disrupt the lattice. A more recent NMR study indicated that at least one additional Ca2+-binding site lies within C2A (31).

The goal of this study was to examine Ca2+- and membrane-induced conformational changes in C2A and to determine which regions of C2A make direct contacts with membranes. The size of the C2A-Ca2+-liposome complex precludes NMR studies to solve its solution structure, and co-crystallization of proteins with membranes has proven difficult. Therefore, we probed the structure of the C2A-Ca2+-lipid complex using fluorescence spectroscopy. Trp reporters were placed throughout C2A, and the sensitivity of indole side chain fluorescence to environment was exploited to monitor local conformational changes and to delineate the lipid binding interface of this domain. Of the five Trp substitution mutants characterized, only the Trp residues that were placed within Ca2+-binding loop 3 of C2A (in positions 231 and 234) exhibited liposome-induced blue shifts in their emission spectra. Membrane-imbedded quenchers provided direct evidence that this loop significantly penetrated into the lipid bilayer. These findings demonstrate that the Ca2+- and lipid-binding sites overlap within C2A and support a Ca2+-bridge model for the assembly of the C2A-Ca2+-membrane complex (44). In this model, Ca2+ makes direct contacts with both anionic headgroups and with C2A. This model accounts for the lower affinity of specific C2-domains for Ca2+ in the absence of lipids (13, 31, 34, 44), since the Ca2+-binding site may not be completely formed. This model is also consistent with molecular modeling studies of membrane-bound phospholipase C-delta 1 (43, 45) and with kinetic studies of cytosolic phospholipase A2 (34).

Structural studies of phospholipase C-delta 1 also indicate that the binding of metal ions results in the extrusion of positively charged side chains. This conformational change results in the widening of the gap between the metal binding jaws such that it becomes large enough to accommodate a phospholipid head group (41). Similar movement of positively charged residues in the C2A-domain of synaptotagmin may also occur (46). The extruded positively charged residues may mediate interactions with nearby anionic lipid head groups. This hypothesis is further supported by the high sensitivity of the C2A-domain-membrane interaction to increasing ionic strength (Fig. 5) and the presence of conserved basic residues in C2-domains that bind phosphatidylserine (42). In addition, an NMR study of C2A demonstrated that Arg-233 and Lys-236 in loop 3, along with Arg-199 in loop 2, made contacts with a recombinant fragment of syntaxin 1A corresponding to residues 1-177 (46). Arg-233 and Lys-236 flank the Trp residue (234) that we have established inserts into membranes. These findings raise the question of whether C2A can bind to syntaxin and to membranes simultaneously. However, issues regarding the interaction between C2A and 1-177 of syntaxin remain to be clarified since domain mapping studies, using GST-syntaxin fusion proteins, demonstrated that residues 194-288 of syntaxin, but not residues 1-193, mediated interactions with synaptotagmin (36, 47). Furthermore, both C2-domains of synaptotagmin are required to form high affinity stable complexes with syntaxin at low micromolar concentrations (19). Thus it is likely that multiple domains mediate syntaxin-synaptotagmin binding.

How might the Ca2+-driven insertion of a Ca2+-binding loop of C2A, into membranes, participate in excitation-secretion coupling? Synaptotagmin is associated with an assembly composed of syntaxin, SNAP-25, and synaptobrevin that is thought to serve as the "core complex" of the synaptic vesicle fusion machinery (24). Association of synaptotagmin with the core complex is potentially mediated via its high affinity Ca2+-independent interaction with SNAP-25 (48). The SNAP-25-synaptobrevin-syntaxin complex appears to zipper together into parallel (49) coiled coils (35, 50), resulting in the very close apposition of the vesicle and target membranes. It is tempting to speculate that the Ca2+-triggered oligomerization of synaptotagmin could result in the clustering of the core complex, thereby forming a collar or spiral-like structure at the vesicle-plasma membrane interface. The Ca2+-triggered penetration of C2A-loop 3 into membranes may function to destabilize the lipid bilayer by inducing lateral phase separations that facilitate bilayer fusion (51). Alternatively, the lipid binding properties of C2A may serve to bring the cytoplasmic domain of the core complex into direct contact with the lipid bilayer. As Ca2+ levels rise, the conformational changes associated with the Ca2+-dependent synaptotagmin-syntaxin interaction may generate the final force or movement that results in bilayer fusion. Interestingly, perforin, a protein secreted from activated cytotoxic T lymphocytes and natural killer cells, is able to bind to membranes in a Ca2+-dependent manner via its C2-domain. Once bound, perforin inserts into membranes where it polymerizes to form large pores. Future studies will determine whether synaptotagmin and the core complex operate by a similar mechanism.

While such models are highly speculative, future studies on the conformational changes in the core complex, induced by binding Ca2+-synaptotagmin, coupled to kinetic studies of Ca2+-synaptotagmin-effector interactions, will help to refine models for the late Ca2+-triggered steps in exocytosis.

    ACKNOWLEDGEMENT

We thank David M. Gaston for verifying the C2A secondary structure and generating the MolScript images used in this manuscript. We also thank Meyer Jackson for critical comments on this manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 56827-01, the Department of Biostatistics and Medical Informatics Computational Biology Resource, and grants to the University of Wisconsin Medical School from the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Physiology, SMI 129, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-263-1762; Fax: 608-265-5512; E-mail: erchapma{at}facstaff.wisc.edu.

1 The abbreviations used are: GST, glutathione S-transferase; C2A, first C2-domain of synaptotagmin; C2B, second C2-domain of synaptotagmin; PS, phosphatidylserine; PC, phosphatidylcholine; PL, phospholipids.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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