Figure 4. Position-dependent effects of layer mutations on SNARE assembly shown by the FECs of the wild-type and mutant SNARE complexes.

The SNARE mutations and their layer numbers are colored based on their associated domains: blue for NTD, green for MD, and red for CTD. While most mutants assemble sequentially as the wild type (WT), some mutants exhibit altered folding pathways, in which two neighboring domains fold and unfold cooperatively as combined domains. Throughout the text, two-state transitions of these new domains are marked by their associated subdomains connected by “_” (Figure 4—figure supplement 1). By comparison, overlapping sequential transitions are indicated by their associated domains linked by “+”. Different folding domains are marked by unique colored ovals or rectangles. Folding energies of the same domain in different SNARE complexes can be compared by the equilibrium forces of the domain transition. The black and gray horizontal lines indicate the average equilibrium forces of CTD (16.5 ± 0.8 pN) and MD+NTD (17.2 ± 0.8 pN), respectively, of the wild-type SNARE complex. CTD, C-terminal domain; FECs, Force-extensioncurves; MD, middle domain; NTD, N-terminal domain.

DOI: http://dx.doi.org/10.7554/eLife.09580.016

Figure 4—figure supplement 1. Comparison of sequential and cooperative domain transitions, using MD and NTD as an example.

The wild type SNARE complex sequentially unfolds in the order of LD, CTD, MD, and NTD and folds in the reverse order, which is caused by the increasing mechanical stabilities of domains from LD to NTD. However, a SNARE mutation may preferentially alter the mechanical stability of the domain in which the mutation resides, changing the order of the domain stabilities. As a consequence, we found that the SNARE mutations often led to combinations of two neighboring domains into new domains that cooperatively folded and unfolded as single units. Throughout the text, these new domains are designated by their associated sub-domains linked by “_”. All neighboring domain combinations were observed, including LD_CTD in SNAP-25 mutant M71A/I192A (Figures 4 and 6), CTD_MD in VAMP2 mutants L60A and L63, and MD_NTD in VAMP2 mutant M46A (Figures 4 and 5). By comparison, two or more sequential transitions in an overlapping force region are designed by their associated domains linked by “+”. For example, the transitions shown in Figure 2A can be indicated by “CTD+MD+NTD”. CTD, C-terminal domain; LD, linker domain; MD, middle domain; NTD, N-terminal domain.

DOI: http://dx.doi.org/10.7554/eLife.09580.017