Figure 2. Molecular determinants of Complexin clamping function.

The effect of mCPX mutants on docking and clamping of spontaneous fusion was assessed using a single-vesicle analysis with a pore-spanning bilayer setup. (A) Inclusion of mCPX increases the number of docked Syt1-vSUVs and this stimulatory effect is greatly reduced when the interaction of the CPX^cen to the SNAREpins is disrupted targeted mutations (mCPX^4A). In contrast, deletion of the N-terminal domain (CPX^26-134) or accessory helix (CPX^48-134) or the c-terminal portion (CPX^26-83) exhibit limited effect of the vesicle docking. In all cases, a mutant form of VAMP2 (VAMP2^4X) which eliminated fusion was used to unambiguously estimate the number of docked vesicles after the 10 min interaction phase. (B) The time between docking and fusion was measured for each docked vesicle and the results for the whole population are presented as a survival curve (Kaplan-Meier plots). Syt1-vSUVs (black curve) are diffusively mobile upon docking and fuse spontaneous with a half-time of ~5 s. Addition of soluble mCPX (red curve) fully arrest fusion to produce stably docked SUVs that attach and remain in place during the entire period of observation. CPX mutants with impaired SNARE interaction (mCPX^4A, green curve) or lacking the accessory helical domain (mCPX^48-134, yellow curve) fail to clamp fusion whilst the removal of c-terminal portion (mCPX^26-83, purple curve) produces a partial clamping phenotype. The N-terminal domain is not involved in establishing the fusion clamp (C) End-point analysis at 10 s post-docking shows that the both the accessory helix deletion (mCPX^48-134) and CPX_cen modifications (mCPX^4A) result in complete loss of inhibitory function and cannot be rescued even at 20 μM concentration. In contrast, the clamping function of the c-terminal deletion mutant (mCPX^26-83) is fully restored at high CPX concentration. The average values and standard deviations from three independent experiments (with ~300 vesicles in total) are shown. **p < 0.01; *** p < 0.001 using the Student’s t-test.

Figure 2—figure supplement 1. Survival analysis (Kaplan-Meier plots) of Syt1-vSUVs shows that the loss of clamping phenotypes observed with CPX mutant with impaired SNARE interaction (mCPX^4A, green curve) or lacking the accessory helical domain (mCPX^48-134, blue curve) is not rescued at high (20 μM) CPX concentrations.

In contrast, increasing the CPX concentration fully-restores the inhibitory function of the c-terminal deletion mutant (mCPX^26-83, purple curve). The average values and standard deviations from three independent experiments (with ~300 vesicles in total) are shown.

Figure 2—figure supplement 2. Dose-dependency analysis using Syt1-vSUVs shows that CPX mutant with a hydrophobic mutation (mCPX^L117W, red curve) designed to improve its membrane association is more efficient in clamping fusion as compared to the CPX^WT (black curve).

This implies that the c-terminal domain contributes to clamping function by increasing the local CPX concentration via membrane interaction. The average values and standard deviations from three to four independent experiments (with ~250 vesicles in total) are shown.