Figure 5. Calcium-induced membrane insertion of Syt1 C2AB hydrophobic loops are critical for both pore nucleation and expansion.

(A) Schematic depiction of Syt1 C2B domain’s calcium-dependent interactions with membranes. Calcium-free C2B interacts with acidic lipids through its poly-lysine motif (highlighted in cyan as in Figure 1). Upon binding to calcium, hydrophobic residues (V304 and I367 on C2B) insert into the membrane, causing C2B to reorient (Chapman, 2008) and inducing membrane curvature (Martens et al., 2007; Hui et al., 2009). In the presence of PI(4,5)P₂, the calcium-bound C2B assumes a tilted conformation with respect to the membrane (Kuo et al., 2011). M173 and F234 on C2A top loops similarly insert into membranes in a calcium-dependent manner, with similar effect on orientation and curvature generation (Chapman, 2008) (not shown). A mutant with the membrane-inserting residues replaced with tryptophans (M173W, F234W, V304W, and I367W, ‘4W’) binds membranes more avidly, resulting in more membrane tubulation activity, whereas alanine substitution of the same residues (‘4A’) abolishes membrane penetration and curvature induction (Martens et al., 2007). (B) Pore nucleation rate (mean ± S.E.M) in the presence of wildtype, 4W and 4A mutants. Student's t-test was used to assess significant differences between the ‘no C2AB’ group and the rest. (C) Mean open-pore conductance (± S.E.M) for the conditions indicated. Two-sample Kolmogorov-Smirnov test was used to assess significant differences between the ‘no C2AB’ group and the rest. (D) Cumulative density functions for mean open-pore conductances for wild-type Syt1 C2AB, 4W and 4A mutants. In A, calcium-free C2B was rendered from PDB 5w5d (Zhou et al., 2017) and calcium-bound C2B was rendered from 5kj7 (Lyubimov et al., 2016). *, **, *** indicate p<0.05, 0.01, and 0.001, respectively.