Fig. 5. The tomosyn SNARE motif binds to the template complex to block its association with SNAP-25.

a Experimental setup to investigate the effect of the tomosyn SNARE motif on the folding of the synaptic template complex Munc18-1:syntaxin-1:VAMP2 (PDB ID 1SFC). b FECs of the VAMP2-Syx conjugate in the presence of 2 µM Munc18-1, 60 nM SNAP-25, and 0 (#1) or 2 µM (#2 and #3) tomosyn SNARE motif in the solution. The presence or absence of these proteins free in solution is indicated by “+” or “−“, respectively. The dashed rectangle and oval mark reversible folding and unfolding transitions of Munc18-1-bound open syntaxin and the template complex, respectively. Red arrows indicate events of SNAP-25 binding to the template complex. Green dashed lines indicate the template complex state (state 7). c Schematic diagrams of different states and their associated energies (E) and transition rates. These states include the fully assembled SNARE complex with or without Munc18-1 bound to the Habc domain of syntaxin (state 1), unfolded SNARE motifs (5), open syntaxin (6), the template complex (7), and the tomosyn-bound template complex (7*). In all these states, Munc18-1 can bind to the N-terminal region of syntaxin-1, including its Habc domain. d Extension-time trajectories at the indicated constant forces (F) showing folding and unfolding transitions between the states labeled on the right. The red curves represent the idealized transitions derived from hidden Markov modeling. The red arrow designates the SNAP-25 binding event. e Extension-time trajectories at constant forces showing folding and unfolding transitions of the Munc18-1-bound open syntaxin-1 in the absence (top) or presence of the tomosyn SNARE motif in the solution. See also Supplementary Fig. 9.