Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly*

Synaptic exocytosis relies on assembly of three SNARE proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE onto a binary SNARE complex on the target plasma membrane. Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE SNAP-25B, which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 kBT, but did not affect N-terminal assembly. Our findings suggest that both mutations impair synaptic exocytosis by destabilizing SNARE assembly. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations.