Correspondence to: Axel T. Brunger, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520. Tel:(203) 432-6143 Fax:(203) 432-6946 E-mail:brunger{at}laplace.csb.yale.edu.
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Abstract |
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Synaptotagmins are synaptic vesicle-associated, phospholipid-binding proteins most commonly associated with Ca+2-dependent exocytotic and Ca+2- independent endocytotic events. Synaptotagmin III is a 63.2-kD member of the synaptotagmin homology group; one of its characteristic properties is the ability to bind divalent cations and accessory proteins promiscuously. In the cytosolic portion of this protein, a flexible sevenamino acid linker joins two homologous C2 domains. The C2A domain binds to phospholipid membranes and other accessory proteins in a divalent cation-dependent fashion. The C2B domain promotes binding to other C2B domains, as well as accessory proteins independent of divalent cations. The 3.2 Å crystal structure of synaptotagmin III, residues 295566, which includes the C2A and C2B domains, exhibits differences in the shape of the Ca+2-binding pocket, the electrostatic surface potential, and the stoichiometry of bound divalent cations for the two domains. These observations may explain the disparate binding properties of the two domains. The C2A and the C2B domains do not interact; synaptotagmin, therefore, covalently links two independent C2 domains, each with potentially different binding partners. A model of synaptotagmin's involvement in Ca+2-dependent regulation of membrane fusion through its interaction with the SNARE complex is presented.
Key Words: SNARE, synaptotagmin, C2 domains, crystallography, calcium-binding protein
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Introduction |
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EARLY characterization of synaptic exocytosis demonstrated that Ca+2 floods into the synapse before the propagation of an action potential (
Synaptotagmin has been characterized in at least eight different isoforms in neurons. Several synaptotagmin isoforms also localize to nonneuronal cells (1 (
In vitro, synaptotagmin interacts with several proteins that are essential for exocytotic and endocytotic processes. In its role as a regulator of synaptic vesicle fusion, the C2B domain of synaptotagmin I binds the SNARE protein SNAP-25 (-SNAP, with a high degree of selectivity (
Although most biochemical studies of synaptotagmin have been carried out with the isolated C2A domain of synaptotagmin I, a few studies suggest that the C2B domain may be responsible for homo- and hetero-oligomerization of synaptotagmin isoforms (Chapman et al., 1996). Synaptotagmin I and synaptotagmin II, III can form homo- and heterodimers in a Ca+2-dependent manner (
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Materials and Methods |
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Cloning and Expression
The cDNA corresponding to the mouse gene of synaptotagmin III C2A-C2B, residues 295569 (33,000 kD), was subcloned into pET28A using the NdeI-HinDIII cloning site. The expression plasmid was transformed into BL21(DE3). The bacteria were grown in a BIOFLO3000 using ECPYM1 media (
Gel Shift Assay
Native gel electrophoresis was carried out using 1015% native Phast (Pharmacia) gels with native buffer strips soaked for 2 h in 0.88 M L-alanine, 0.25 M Tris, pH 8.8, and 1 mM EDTA before running the gel. Samples were mixed and allowed to incubate for at least 1 h in 20 mM Hepes, pH 7.8, 150 mM NaCl, 5 mM DTT, and 1 mM EDTA before loading the gel. The concentration of both proteins was determined by amino acid analysis (Keck facility, Yale University). SNARE core complex was expressed in a polycistronic T7 expression vector containing rat synaptobrevin-II, residues 196, rat syntaxin-1A, residues 180262, rat SNAP-25A residues 183 and residues 130206, and purified according to previously published methods (
Purification and Crystallization
30 g of frozen bacterial cells were resuspended in 125 ml of extraction buffer (100 mM sodium phosphate, pH 8.0, 200 mM NaCl, and 10% ethylene glycol) with 200 mg of lysozyme and 0.5% Triton X-100. Passing the lysate twice through a pneumatic cell cracker (Microfluidics) at 10,000 lb/in2 facilitated cell lysis. The histidine-tagged protein was incubated in batch with NTA-Ni (Qiagen) resin overnight at 4°C while slowly mixing. The resin was washed in a column with extraction buffer including 50 mM imidazole until the OD280 was below 0.05. The tagged protein was eluted with 300 mM imidazole. At this point, 10% ethylene glycol, and 5 mM CaCl2 were added to the protein solution. Under these conditions, synaptotagmin III precipitated and was removed by centrifugation in a GSA rotor at 10,000 rpm for 20 min. The protein pellet was washed in 25 mM ethanolamine, pH 8.5, 300 mM NaCl, and 10% ethylene glycol. The protein was resuspended in 30 ml of 50 mM sodium phosphate, pH 7.8, and 5 mM EDTA. The histidine tag was removed by cleaving the protein with TEV protease (BRL) overnight at 4°C. The resuspended protein was filtered through a 0.22-µm filter before ion exchange chromatography with a Mono Q 5/5 (Pharmacia) column in 25 mM ethanolamine, pH 8.0. The purified protein eluted from a 01-M NaCl gradient at 200 mM NaCl. The protein was concentrated to 40 mg/ml. Synaptotagmin III C2A-C2B crystallized in 1.5 M MgCl2 and 100 mM MES, pH 6.5, at 20°C using the hanging drop method. Large hexagonal crystals grew after ~1 wk.
Data Collection
Analysis of the systematic absences in the diffraction data narrowed possible space groups choices to either P6122 or P6222: a = 126 Å, b = 126 Å, c = 118 Å. Inspection of the molecular replacement solution of the C2A domain uniquely determined P6222. The Matthews coefficient calculation predicted two molecules in the asymmetric unit given average solvent content; however, only one protein molecule was found resulting in a solvent content of 70%. Native and trimethyl lead acetate (TMLA) data were collected at station BL1-5 at SSRL at a temperature of 20°C. Diffraction data from several crystals were merged to obtain highly redundant and complete native and anomalous derivative data sets. Diffraction data of the derivative were collected at a wavelength of 1.00 Å to take advantage of the lead (Pb) anomalous signal. Diffraction data were integrated and scaled using the DENZO package (
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Structure Solution
Placement of each C2 domain within the asymmetric unit was attempted by molecular replacement as implemented in CNS (
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Partial Disorder of the C2B Domain
The program O was used to build the initial model (-helix between the between strands ß7 and ß8 of the C2B domain was constructed from the available electron density.
Refinement
The model phases were used as prior phase probability distributions to feed back into the heavy atom model refinement resulting in significantly improved SIRAS phases. The MLHL target function (sol) of 0.27 e-/Å3 and a B-factor of 25 Å2. The final model had 98.7% of all residues in allowed regions. Three residues in the C2B domain are reported as disallowed (Ala 553, Lys 557, and Ser 475). Due to the disorder in a portion of the C2B domain, several side chain rotamers could not be absolutely determined and were set to those found in common with the PKC-ß (1a25) and the synaptotagmin I (1rsy) C2 domains. It should be noted, however, that the side chain rotamers in the Ca+2-binding region of the C2B domain could be unambiguously assigned (see Figure 3 A). The Mg+2 and the sulfate ions were included in the model after the protein tracing was completed. It should be noted that at this resolution one cannot exclude the possibility that some of the Mg+2 electron density peaks correspond to ordered water molecules. However, the observed Mg+2 sites correspond to known sites in the divalent cation-binding sites of other C2 domains. The last few COOH-terminal residues of synaptotagmin III are predicted to be
-helical by secondary structure prediction (
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Results |
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Overall Topology
The cytosolic portion of synaptotagmin III contains two tandem C2 domains joined by a short seven-residue linker (Figure 1). Each of the two homologous C2 domains consists of an eight-stranded Greek key ß-sandwich with type I (S-type) C2 topology (-ß bulges (
The Ca+2-binding regions of both C2 domains are directed toward each other in the crystal structure. Only ~300 Å2 of surface area is shared between the two domains, which is likely too small to represent an important interaction in solution. It is therefore unlikely that this contact has physiological function. Thus, we predict that the two C2 domains are largely independent of each other in solution. The Ca+2-binding loop 3 of C2B is disulfide linked to another C2B domain that is related by crystallographic symmetry to another molecule. This covalent linkage does not affect the overall loop conformation since the Ca+2-binding loops of the superimposed C2A and C2B domains are very similar.
The relative orientation of the two C2 domains (Figure 1) represents the conformation of synaptotagmin III C2A-C2B favored by crystallization. The observed partial disorder of the C2B domain in this crystal form suggests that the relative orientation and position of the two C2 domains is variable in solution. This anisotropic disorder is confined to the plane of the ß-sandwich, and the electron density of the ß-sheets of the C2B domain is somewhat smeared out. However, the electron density for the C2B domain at the apex of the fold, including the Ca+2-binding pocket, is well defined, allowing sidechain interpretation of the Ca+2-binding region of the C2B domain (see Figure 3 A).
The C2A and C2B domains are structurally similar with the exception of the -helix between the strands ß7 and ß8 of each C2 domain (Figure 1 and Figure 2). The seven-residue
-helix of the C2B domain is not present in either the C2A domain of synaptotagmin I, the isolated C2 domain of PKC-ß, or the type-II (P-type) C2 domain of PLC-
1. This conserved
-helical insertion has also been reported in the isolated C2B domain of rabphilin-3A (
atoms is 1.5Å.
As in the structure of the C2A domain of synaptotagmin I and the isolated C2 domain from protein kinase C, the C2A domain of synaptotagmin III contains a cis-proline (Pro 411) that precedes the ß-strand containing the polybasic region. The homologous region in C2B, however, does not contain this proline. Instead, it uses a non-proline ß-turn to accommodate the residues leading into the polybasic strand. In the C2B domain, this insertion shifts the polybasic region to a more central location on strand ß4.
Ca+2-Binding Pockets
Synaptotagmin III crystallized in the presence of 1.5 M MgSO4. Although Mg+2 could potentially mimic Ca+2 binding, the aspartate residues in the C2A domain that have been shown by solution NMR and x-ray crystallography to pivot upon divalent cation binding, are not in the Ca+2-bound conformation (Figure 3 A). Under these crystallization conditions, the synaptotagmin III C2A domain is in the unliganded conformation. However, three peaks in electron density difference maps were found in the Ca+2-binding pocket of the C2A domain. These peaks coincide with the positions of the three calcium ions in the PKC-ß C2 structure and were interpreted as Mg+2. The Mg+2 could be responding to the negative electrostatic field from the aspartate residues within the divalent cation-binding pocket of the C2A domain without proper coordination of the aspartate residues.
In the C2B domain, significant electron density, consisting of a single 5sigma}"> peak, was also found in the vicinity of the Ca+2-binding region (Figure 3 A). This peak corresponds to the high affinity Ca+2 site observed in the crystal structures of the synaptotagmin I C2A, the PKC-ß C2, and the PLC-
1 C2 domains. Although the divalent cation coordinating aspartate residues are present in the C2B domain, only one Mg+2 binds in the divalent cation-binding pocket, despite the very high Mg+2 concentration used for crystallization. The interpretation of this electron density peak in terms of a divalent cation-binding site is supported by the observed substitution of the site by a trimethyl lead ion in the TMLA derivative.
The Inter-Domain Linker
The linker between C2 domains for most of the synaptotagmin isoforms, including synaptotagmin III, is seven to nine residues in length (Figure 2). The amino acid composition of synaptotagmin III suggests a rather flexible linkage with two contiguous glycine residues. The primary sequence of the linker is not conserved among the synaptotagmin homology group (Figure 2). The inter-domain linker may be more rigid for the synaptotagmin isoforms that have fewer glycine residues and more proline residues, such as synaptotagmin VII or synaptotagmin VIII (Figure 2). The rigidity of the inter-domain linker in some synaptotagmin isoforms may position the C2 domains for docking to vesicle-fusion related protein complexes, or otherwise restrict the range of motion possible between the two C2 domains. Other tandem C2 domain containing proteins such as rabphilin (
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Discussion |
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Structural Implications for Biochemically Distinct C2 Domains
Synaptotagmin III is characterized by promiscuous binding to various accessory proteins and membrane components (Li et al., 1995). The two C2 domains of synaptotagmin have different binding partners and binding affinities. The x-ray crystal structure of the cytosolic domain of synaptotagmin III provides a structural explanation of these disparities between the two C2 domains. Despite the lack of Ca+2 in the crystallization condition, this structure could still mimic some of the Ca+2-binding properties of C2B domains in the synaptotagmin homology group. In the crystal structure, the C2B domain associates with only one Mg+2. The reduced divalent cation-binding capacity of the C2B domain would leave this area of the molecule with a residual negative charge relative to the C2A domain. This difference between the C2A and C2B domains may explain some of the biochemical differences observed in in vitro experiments. Overall, the C2A domain of synaptotagmin III has a more uniform electrostatic surface potential than the C2B domain (Figure 4). The surface of the C2B domain possesses distinctly basic and acidic areas (Figure 4). These charged areas may be important for the Ca+2-independent interactions observed in the isolated C2B domain.
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The Ca+2-binding pocket of the C2B domain is chemically similar to the Ca+2-binding pocket of the C2A domain; however, the shape of the pocket is very different (Figure 3 B). In principle, this can be explained by either a difference in backbone conformation between the two domains or by a difference in sidechain packing. Superposition of the two domains does not reveal a significant deviation in the backbone position; however, one cannot rule out more subtle backbone differences within the coordinate error of the crystal structure. Although synaptotagmin III has been implicated in Mg+2-dependent phospholipid binding (
A sulfate ion from the crystallization medium is located near Lys 356 of the C2A domain of synaptotagmin III. This ion may mimic phospholipid binding to synaptotagmin, since this face of the C2A domain interacts with the phospholipid bilayer (
Model of C2B-mediated Oligomerization
Only one molecule is present in the asymmetric unit of this crystal form. The crystallographic symmetry together with primary sequence analysis of the available synaptotagmin isoforms may provide clues to the mechanism of C2B domain homo- and heterodimerization. Two crystallographically related molecules in this hexagonal crystal form direct their divalent cation-binding pockets toward the sixfold crystallographic axis. The resulting crystal packing contacts include the sequence Asp-Phe-Asp (386388), which includes two of the divalent cation-binding residues in C2 domains. These three residues are conserved in all C2A domains with the exception of synaptotagmin VII. In the C2B domain, the homologous motif is Asp-Tyr-Asp (520522), with few exceptions among the other synaptotagmin isoforms. In the crystal structure of the synaptotagmin III C2A-C2B domains, an alternate rotamer of Tyr 521 can be modeled to coordinate Asp 466 of the crystallographically related molecule to form a hydrogen bond. Aspartate 466 anchors the divalent cation-binding chain in the Ca+2-binding pocket of C2 domains. This putative interaction may provide an initial nucleation point for self-association. The sequence variability present in the other isoforms of synaptotagmin, for example, synaptotagmin VII, VIII, and XI, may modulate their individual binding properties to other synaptotagmin isoforms.
Interactions of Synaptotagmin with the SNARE Complex
The core of the SNARE complex, composed of synaptobrevin-II (196), syntaxin-1A (180262), SNAP-25A (183), and SNAP-25A (130206), interacts with the C2A and C2B domains of synaptotagmin III independent of divalent cations (Figure 5). We have modeled the association between these two moieties using the following arguments: first, the position of the presynaptic membrane restricts the possible contacts between the two C2 domains of synaptotagmin and the SNARE complex. The syntaxin component of the SNARE complex embeds its transmembrane -helix into the presynaptic membrane; likewise, the synaptobrevin transmembrane
-helix is anchored in the vesicle phospholipid membrane. Our model, therefore, puts the synaptobrevin
-helix on top of the complex and the syntaxin
-helix on the bottom relative to the presynaptic membrane bilayer on the bottom of Figure 6. The palmitoylated SNAP-25 linker would also contact the presynaptic membrane, so it localizes to the membrane associated with syntaxin. Second, the divalent cation-binding loops (CalB) of the C2 domains of PLA2 contact phospholipid bilayer with some residues embedded in the bilayer itself (
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Specific regions of the SNARE complex are likely to be involved in synaptotagmin C2 domain interactions. The NH2-terminal domain of syntaxin interacts with the C2A domain of synaptotagmin I (
According to the zipper model of SNARE-mediated fusion (
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Acknowledgements |
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The authors thank P.D. Adams for stimulating discussions and H. Bellamy for assistance with data collection at SSRL BL1-5. We would like to thank Pat Fleming for help with the packing analysis of synaptotagmin III, Thomas Südhof for the initial clone synaptotagmin III, Josep Rizo for the help with the correct alignment of C2B domains, Mark Bowen for helpful discussion, Mike Reese and Steve Kaiser for preparing the fusion complex, and Stephen Sprang who provided a Welsh grant (I-1229) for early work on synaptotagmin III.
The SSRL Biotechnology Program is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program and the Department of Energy, Office of Biological and Environmental Research. J.A. Ernst is supported by a pre-doctoral fellowship in biophysics from the National Science Foundation. Coordinates will be deposited with the PDB and are also at http://atb.csb. yale.edu.
Submitted: 2 August 1999
Revised: 22 September 1999
Accepted: 30 September 1999
1.used in this paper: PKC, protein kinase C; SNARE, soluble N-ethylmaleimidesensitive factor attachment protein receptor; SIRAS, single isomorphous replacement, anomalous scattering; SytI, synaptotagmin I; TMLA, trimethyl lead acetate
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References |
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