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. 1999 Nov 1;147(3):589-98.
doi: 10.1083/jcb.147.3.589.

Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction

R B Sutton  1 , J A Ernst, A T Brunger
Affiliations

Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction

R B Sutton et al. J Cell Biol. .

Abstract

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 seven-amino 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 A crystal structure of synaptotagmin III, residues 295-566, 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.

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Figures

Figure 1
Figure 1
Overall structure of the cytosolic domain of synaptotagmin III. The gold arrows correspond to β-strands, the blue corresponds to α-helices, and the red spheres to Mg+2.
Figure 2
Figure 2
Sequence alignment of the synaptotagmin homology group with ClustalW: yellow arrows correspond to β-strands in the C2A domain; gold arrows correspond to β-strands in the C2B domain; green waves correspond to the flexible linker. Blue helices correspond to α-helices. Syt3, mouse synaptotagmin III (294–588) (Swiss-Prot: SYT3_MOUSE); Syt1, mouse synaptotagmin I (271–421) (Swiss-Prot: SYT1_MOUSE); Syt4, rat synaptotagmin IV (152–425) (Swiss-Prot: SYT4_RAT); Syt5, rat synaptotagmin V (105–386) (Swiss-Prot: SYT3_RAT); Syt6, rat synaptotagmin VI (226–511) (GenBank: U2O105); Syt7, rat synaptotagmin VII (129–403) (GenBank: U20106); Syt8, mouse synaptotagmin VIII (61–355); Syt11, rat synaptotagmin XI (152–430) (GenBank:AF000432); rab3A, rat rabphilin-3a (383–684) (SWISS-PROT: RP3A_RAT); doc2, mouse doc2 (125–412) (PIR: JC4921).
Figure 3
Figure 3
(A) Experimental electron density map using native amplitudes and density modified SIRAS phases of the C2A and C2B Ca+2-binding pockets contoured at 1.5 σ. Some of the coordinating aspartic acid residues are shown as balls-and-sticks. The labels for the Mg+2 sites follow the numbering convention of the C2 domain of PLC-δ1 (Essen et al. 1997). (B) Connelly surface obtained by MidasPlus of synaptotagmin III C2A and C2B using a probe radius of 1.4 Å. Arrows point to the divalent binding pockets in the C2 domains.
Figure 3
Figure 3
(A) Experimental electron density map using native amplitudes and density modified SIRAS phases of the C2A and C2B Ca+2-binding pockets contoured at 1.5 σ. Some of the coordinating aspartic acid residues are shown as balls-and-sticks. The labels for the Mg+2 sites follow the numbering convention of the C2 domain of PLC-δ1 (Essen et al. 1997). (B) Connelly surface obtained by MidasPlus of synaptotagmin III C2A and C2B using a probe radius of 1.4 Å. Arrows point to the divalent binding pockets in the C2 domains.
Figure 4
Figure 4
Surface plot showing the electrostatic potential of synaptotagmin. Blue, positive; red, negative charge. Charges were obtained from the OPLS force field. The electrostatic surface was contoured between −10kT/e and +10kT/e. Figure prepared with GRASP (Nicholls et al. 1991).
Figure 5
Figure 5
Ca+2-independent synaptotagmin-SNARE complex interaction: 10–15% native Phast gel (Pharmacia) run in the presence of 1 mM EDTA. Lanes B–F contain 1.6 μg of core SNARE complex; lanes A–E contain 3.2, 3.2, 2.4, 1.6, and 0.8 μg of synaptotagmin III C2AB.
Figure 6
Figure 6
Cartoon illustrating the proposed association between synaptotagmin (gold colored protein) and the SNARE fusion complex (SNAP25, green helix; syntaxin, red helices; and synaptobrevin, blue helix) and the interaction with the phospholipid membrane (yellow plane). Ca+2 are illustrated as red spheres. The transmembrane anchor of syntaxin and the linker between the H1-H2 domain (red helices to the left of C2A) and the H3 domain of syntaxin (red helices in the SNARE bundle) have been omitted for clarity. In our model, we predict that the Ca+2-binding sites of synaptotagmin interact with the presynaptic membrane. The interactions between the SNARE complex and synaptotagmin probably involve the COOH-terminal (membrane-proximal) portion of the SNARE core complex (Kee and Scheller 1996) and the cup-shaped, polybasic regions of the synaptotagmin C2 domains. The association between the NH2-terminal (H1-H2) domain of syntaxin (red helices to the left of the C2A domain) and the C2A domain of synaptotagmin was modeled according to NMR data (Shao et al. 1997). The peptide distance between the transmembrane domain and the SNARE-binding domain of synaptobrevin (blue helix) is exaggerated in this cartoon.
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