however this oligomerization was enhanced by Ca2+ (Figure 4B, n = 3, p,0.05). The truncated GST-C2B fusion proteins (GST- DK and GST-DW) were also used in self-association experiments with full length MBP-C2B. Consistent with previous findings [37], the GST-DK construct did not bind to the wild-type MBP-C2B (Figure 4A, n = 3, p,0.01). Under the same reaction conditions however, the GST-DW showed an increased capacity to bind MBP-C2B both in the presence Ca2+ (fourfold; Figure 4A, n = 3, p,0.01) and in EGTA (data not shown). Changes in the level of oligomerization caused by addition of Ca2+ or in using the C- terminal truncation constructs, are similar to the changes seen in the binding of the STNA/STNB proteins. It seems likely, therefore, that the increase in binding of MBP-STNA and MBP- STNB to the C2B domain deletions is due to the increase in the ability of these deletions to oligomerize, and that the Ca2+ enhancement of STNA and STNB binding to the C2B domain is also due to the Ca2+-enhanced oligomerization of the C2B domain. Mutation of the Ca2+-binding region reduces STNA and STNB binding If the binding of STNA and STNB to the SYT-I C2B is truly dependent on the Ca2+-dependent oligomerization of the SYT-I C2B domain then if the C2B Ca+ binding were to be specifically ablated, then one might expect that this would also lead to a reduction in the binding of both STA and STNB. We therefore asked if the binding of the Stoned proteins is altered in the D3,4N mutant of Syt-I C2B. The D3,4N mutant, replaces two aspartate residues, D416 and D418, with asparagines. Both aspartates are involved in the binding of two Ca2+ ions to the C2B domain within the loops [38]. These mutations were reported to reduce the effect of Ca2+ on C2A-C2B oligomerization and caused the mutant protein to oligomerize in the absence of Ca2+ [37]. The RMSD of the D3,4N structures during 100 ps MD simulation at 300 K suggested that this mutant has similar structural integrity to the full-length C2B (Figure S2). Using a combination of MBP-D3,4N and GST-D3,4N, we confirm that the D3,4N mutant does abolish the Ca2+-enhanced C2B domain self-association (Figure 4B, n = 3, p,0.05. As well as reducing the Ca2+-dependent self-association of the C2B domain, the D3,4N mutant also reduces both STNA and STNB binding (Figure 4C). This then confirms that the Ca2+-induced binding of both STNA and STNB to the SYT-I C2B domain is primarily due to the Ca2+-induced self-association of the C2B domain. STNB interacts with the Ca2+-binding loop of the SYT-1- C2B domain Since the D3,4N mutant reduces the binding of the STNB protein to the C2B even in the absence of Ca2+, we investigated the possibility that the STNB protein might directly interact with this region of the C2B domain. Competition experiments were performed using a peptide, pC2B1. This peptide covers amino acid residues 407–424 and includes all of the residues of loop 3 and b-strand 6 [38,39]. Addition of the pC2B1 peptide out- competed MBP-STNB in a dose dependent manner (Figure 5A and 5B, n = 3). Whereas the peptide also reduced, but did not eliminate, the binding of MBP-STNA to GST-C2B (Figure 5A and 5B, n = 3). At 4 mM, pC2B1 abolishes MBP-STNB (0.25 mM) binding to 1 mM GST-C2B. By comparison MBP-STNA binding is reduced but only by 60% (n = 3, p,0.05). When tested for its effects on self-association of the C2B domain, the pC2B1 peptide also reduced C2B oligomerization, but only to an extent similar to the reduction observed for MBP- Interaction between Stoned and Synaptotagmin-1 STNA binding (Figure 5C). At 2 mM, pC2B1 reduces MBP-C2B (0.25 mM) binding to 1 mM GST-C2B by approximately 40%, thus pC2B1 significantly reduces but does not eliminate the self- association of the C2B domain. This would suggest that the partial inhibition of STNA binding was most likely due to the effect of the pC2B1 peptide on oligomerization, while the total inhibition of STNB binding can only be partially due to the failure of the C2B protein to oligomerize in the presence of the pC2B1 peptide. If, however, STNB were to interact directly with residues in the loop, then perhaps the peptide might be competing directly with STNB. Two other peptides were tested in these assays (Figure S3). The first was a random peptide (pCTRL). This peptide had no effect on the binding of STNB. A second peptide (pC2B1.M) was equivalent to pC2B1 but with the tyrosine at position 417 in the C2B domain substituted with alanine. As tyrosine-containing motifs are known binding sites for the m-subunit of AP-2, it was hypothesized that such a mutation might alter STNB binding. This Y417A mutant peptide (pC2B.M) behaved in exactly the same way as the wild-type peptide indicating that this tyrosine is not important for STNB binding. Although pC2B1 acts to reduce the oligomerization of the C2B domain, and hence the binding of the Stoned proteins, these data suggest that there is a direct interaction between binding of the loop region of the SYT-1-C2B domain and to the mHD of STNB. Together with the D3,4N binding studies, STNB may bind to the oligomerized C2B domains in the vicinity of the Ca2+ binding loop. Discussion In the present study, we have investigated the effect of Ca2+ upon Stoned proteins binding to SYT-1 C2B domain. We had reported that the Drosophila mHD of STNB binds to C2B domain of SYT-1 [17], which is in agreement with Martina et al. that showed the mHD of the stonin-2, the mammalian homologue of the Drosophila STNB protein, could also bind SYT-1 [35]. Jung et al. have reported that stonin-2 is able to bind the C2A domain, even though the role of Ca2+ in this binding was not explored [40]. We also observed that STNB is able to bind to the C2A domain in vitro, albeit at a much lower level as compared to the binding to the C2B domain. Our data show that the binding of Drosophila STNB to Drosophila SYT-1 is different from that of the murine m2 subunit of AP-2. While m2 binding to C2B domain requires PS and Ca2+ [20], the binding of the STNA and STNB proteins did not require PS (Figure 2). In fact PS–containing phospholipids nearly abolished the STNB binding to SYT-1 C2B (Figure 2B). Deletion of the polyK region of the C2B domain also did not abolished STNB binding to SYT-1 (Figure 3C), indicating that this region cannot be the binding site for either STNA or STNB. Our data support a recent publication that showed that, in Drosophila, m2 cannot replace the function of the mHD of STNB in vivo [41], and suggest that STNB and AP-2 might represent alternative mechanisms for synaptic vesicle recycling. AP-2 is ubiquitously expressed and implicated in general mechanisms of endocytosis from the plasma membrane, while in Drosophila, STNB is expressed and functions only in the nervous system [42]. Differences in the mHD of STNB and the m2 domain of AP-2 (this study, [20] and [41]) may reflect the need for flexibility of m2 subunit of AP-2 to act in a number of endocytic pathways, while the function of STNB may be specific for synaptic vesicle retrieval. Our data, in conjunction with a report that STNB may specifically regulate the sorting of a subset of SV [41], suggest the Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound SYT-1 associated SV. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38822