fusion. In a recent paper [24], it is shown that SYT-1 bound to PtdIns at the same level as PS and that this binding is also required Ca2+; while the binding of PIP2 with SYT-1 is less affected by Ca2+. It would be interesting to investigate whether these lipids will have similar effect as PS in affecting Stoned and C2B SYT-1 binding. The role of STNA in the synaptic vesicle cycle remains elusive. The presence of STNA at the larval NMJ appears nonessential [46]. However STNA is certainly associated with synaptic vesicles and it is clear that STNA has a strong affinity for SYT-1 [17]. We have shown that the oligomerization of SYT-1 dramatically increases the binding of STNA and presumably STNA will bind under similar in vivo conditions as STNB. The presence of putative AP-2 binding motifs in STNA [29], may make vesicles bound by STNA targets for AP-2 mediated endocytosis. This is in contrast to STNB that lacks such AP-2 binding motifs. Whatever the specific mechanism of action of the STNA protein, it is clear from this study that the action of Ca2+ has a marked effect on STNA and STNB association with SYT-1 and confirms them as important proteins in the mechanism(s) of synaptic vesicle recycling in Drosophila. Materials and Methods Recombinant proteins SYT-1 C2A and C2B constructs were generated in the pGEX- 4T-1 vector and expressed as GST-tagged proteins in E. coli. The C2A construct (bases 137-326) was produced as described previously [17]. The C2B wild-type construct (bases 323–474) was produced by PCR from the Drosophila syt cDNA clone using a 59 primer that generated an EcoR1 site (59CTGGTCAGCGT- TGCCTTCGAGGGCGG39) and the 39 primer that introduced a terminal Xho1 site as well as an internal BglII site but retained the correct amino acid sequence (59CTCGA-GTTACT- TAATGTTCTTCAAGATCTCGTC39). The DW construct was produced using the same 59 primer as described above and the 39 primer (59CTCG-AGTTACTGGGC- GATGGGCCGGCG-39) that again inserted a terminal XhoI site but also introduced a UAA termination codon that would terminate translation of the C2B domain immediately prior to the WHTL motif. The GST-DH was generated by PCR using the same 59 primer and 39primer (59CTCGAGTCCGG- TGCCCATGCAGCCA39) again inserted a terminal XhoI site but also introduced a UAA termination codon that would delete helix A, helix B and b-strand 8 of the C2B domain. The GST-DK was generated using 59 primer (59GAATTCTGCACCCT- CAACCCTTACTA39) and 39 primer (59CTCGAGTTACTT- CAT-GTTCTTCGCGATCTCGTC) to amplify residue C386 to K474. The GST-D3,4N was created by two step PCR amplifi- cation using pGEX/C2B as template DNA. The 59 end was amplified using primer 59 primer (59ACCGTCGTGAACTA- CAACCGTATTGGCAACCTCC39) and 39 primer (59GAGCTGCATGTGTCAGAGG39). The 39 end of GST- D3,4N was amplified using 39 primer (59GGAGGTGCCAA- TACGGTTGTAGTTCACGACGGT39) and 59 primer (59CCAGCAAGTATATAGCATG39). Overlapping PCR was used to generate the full length D3,4N construct using 59 primer (59CCAGCAAGTATATAGCATG39) and 39 primer (59GAGCTGCATGTGTCAGAGG39). The m-homology domain (mHD) of STNB was amplified using 39 primer (59CTCGAG- CAGCACCTAGCCAACTTA39) and 59 primer (59GAATTCCGA-GAGCGAGCGTTGACA39). The DNA se- quences encoding the coiled-coil domain (CCD) of STNA and the mHD of STNB were inserted into the pMAL-c2x vector, and Interaction between Stoned and Synaptotagmin-1 expressed in E. coli as MBP tagged proteins as previously described [17]. All constructs were verified by sequencing. For the C2B self association assay, the DNA encoding the C2B domain was also inserted into pMAL-c2X, and expressed in E. coli to generate MBP-C2B. Protein expression and purification MBP and GST fusion proteins were produced from cultures according to the manufacturers’ instructions (NEB and Amer- sham), and bound to amylose resin (MBP fusions) or glutathione resin (GST fusions). To remove contaminating nucleic acids present in the E. coli lysate and known to alter the properties of recombinant C2B, GST-C2B and MBP-C2B fusion proteins bound to resin were washed twice in 10x volume of 20 mM MES pH 6.3, 20 mM CaCl2, and 0.6 M NaCl (1h on rotating wheel at 4uC), 10x volume of cold TBS, 10x volume of Tris-CaCl2 buffer (1h on rotating wheel at 4uC) and followed by 10x volume of cold TBS, adapted from Ubach et al [47]. The purity of expressed proteins was determined by measuring the A280/260 [20] and analyzed by Coomassie stained SDS PAGE gel. Molecular Dynamics simulation The three dimensional model structures of C2B and its variants (C2B, D3,4N, DH and DW) were generated using the 3D- JIGSAW comparative modeling server (http://www.bmm.icnet. uk/˜ 3djigsaw/) using the NMR structure of the C2B domain of rat Synaptotagmin-1 (PDB ID 1tjx) as template [38]. Molecular Dynamics (MD) simulation was performed using the program NAMD [48] on a dual-core machine (2.6 GHz) loaded with MS Windows. The CHARMM22 force field was used for the simulation. The input files were prepared using the program VEGA ZZ [49]. The PDB files C2B and its variants were modified by adding hydrogen and solvating in a water box that leaves a 0.2 nm space around the solute. Energy minimization was done for 5000 steps using the steepest descents method. MD simulation began by heating the system from 0 to 300 K for 3 ps, and was followed by an MD simulation at 300K for 100ps timescale using an integration time step of 1 fs. Nonbonded interactions were evaluated with a cutoff distance of 12.0 A˚ and a switch distance of 2.0 A˚. Langevin dynamics were used during MD simulation, which was done without constraining any degrees of freedom. MD parameters and structures were written every 1ps. The root mean square deviation (RMSD) of the structures during simulation was analyzed using the program VMD [50]. Protein-protein interaction experiments Pull down assays were conducted for 2 h at 4uC using 1.5 mM purified GST proteins as bait on 50 ml glutathione resin and 0.25 mM MBP-STNA and MBP-STNB proteins as the prey protein. The pull downs were carried out on a rotating wheel, for 2 h at 4uC, in a total volume of 500 ml of 10 mM HEPES buffer pH 7.4, 1 mM MgCl2, 1 mg/ml BSA containing either 1 mM CaCl2 (Ca2+ binding buffer) or 1mM EGTA (EGTA binding buffer). The beads were then collected by centrifugation, and washed twice with 1ml of binding buffer, followed by 1 ml of cold TBS. The complex of MBP-STNA/STNB and GST-C2B was eluted with 20mM glutathione and analyzed by Western Blot using anti-MBP antibody (New England Biolabs) using Amersham ECLTM Western Blotting System. The amount of MBP-STNA/ STNB proteins was then quantified by densitometry using ImageJ or Fuji MacBas software programs and the measured intensity was normalized according to the eluted concentration of GST-SYT proteins visualized by Ponceau S stain. The quantified band density was then normalized to value of binding of respective PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e38822