Figure 5: Munc13–1 recruits SNAP25 to Munc18–1/Syntaxin-1/VAMP2 template complex.

(A) Experimental setup for optical tweezers. A single SNARE complex was pulled from the C termini of Syntaxin 1A (red) and VAMP2 (blue) via two DNA handles attached to two optically trapped polystyrene beads. The N-termini of Syntaxin 1A and VAMP2 were cross-linked via a disulfide bond. Munc18–1 (gray) and the MUN domain of Munc13–1 (yellow) were added in the solution. (B) Representative force extension curves (FECs) obtained for SNARE complexes assembled with SNAP25^WT or SNAP25^G4S in the presence of Munc18–1 and MUN domain as indicated. The Syntaxin–VAMPs conjugate was pulled (grey FECs) or relaxed (black FECs) by changing the separation between two optical traps at a speed of 10 nm/s or held at constant mean force around 5 pN (red FECs). The molecular states associated with different FEC regions are indicated by the corresponding state numbers (see Supplementary Figure 5 for their molecular diagrams) (C) Probability of full SNARE complex assembly from the Munc18–1/Syntaxin-1/VAMP2 template complex observed within 100s at 5 pN constant mean force. MUN domain ability to stimulate SNARE complex assembly is significantly diminished with the SNAP25 linker domain mutant (SNAP25^G4S) as compared to SNAP25^WT (p = 0.065). The N value refers to the total number of trials or pulling/relaxation rounds and average and standard error of the means are shown the error bar indicates the standard deviation (σ) of the corresponding probability (p) calculated as σ = √(p(1−p)/N).