Ca² dependent oligomerization regulates STNA and STNB proteins binding to the C2B domain of SYT-1.

<p><b>A.</b> Truncation of C2B affects oligomerization. The N-terminal polylysine deletion (GST-ΔK) reduced MBP-C2B binding while the C-terminal tail deletion (GST-ΔW) increased MBP-C2B binding fourfold (n=3) in comparison with binding to GST-C2B. Independent experiments were done in Ca²⁺ buffer. The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-C2B to GST-ΔK or GST-ΔW is normalized against MBP-C2B bound to GST-C2B. Each value represents mean ± S.E.M. <b>B.</b> The D3,4N mutation abolished the effect of Ca² to regulate C2B oligomerization in comparison to GST-C2B in Ca² buffer (n=3, p<0.05). The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-C2B to GST-C2B or GST-D3,4N is normalized against MBP-C2B bound to GST-C2B in EGTA buffer. Each value represents mean ± S.E.M. <b>C.</b> The D3,4N mutation also reduced STNA (*n=3, p<0.05) and STNB (**n=5, p<0.01) binding by comparison with binding to GST-C2B in Ca² buffer. In the presence of Ca²⁺ buffer, the D3,4N mutation significantly increased the MBP-STNB binding in comparison to GST-D3,4N in EGTA buffer (***n=5, p<0.05). The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-Stoned proteins to GST-C2B or GST-D3,4N is normalized against MBP-Stoned proteins bound to GST-C2B in EGTA buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot image shows the MBP tagged proteins bound to GST tagged proteins and detected by anti-MBP antibody; while the lower panel is the Ponceau Red staining of GST proteins of the respective blots as loading control.