Guiding AlphaFold to predict how Munc13‐1 opens Syntaxin‐1

The SNARE proteins syntaxin‐1, synaptobrevin, and SNAP‐25 mediate neurotransmitter release by forming SNARE complexes that fuse synaptic vesicles with the plasma membrane. SNARE complex assembly is orchestrated by Munc18‐1 and Munc13‐1 through a highly regulated pathway that starts with syntaxin‐1 folded into a closed conformation and bound to Munc18‐1. It is well‐established that Munc13‐1 opens syntaxin‐1, likely acting catalytically, and that this step is crucial for neurotransmitter release. However, the underlying molecular mechanism remains unknown because it is difficult to obtain structural information on Munc13‐1‐syntaxin‐1 interactions experimentally. Initial attempts with AlphaFold using the syntaxin‐1 cytoplasmic region yielded structures of Munc13‐1‐syntaxin‐1 complexes but syntaxin‐1 remained closed. Interestingly, when using a shorter syntaxin‐1 fragment designed to destabilize the closed conformation, AlphaFold generated a model of Munc13‐1 bound to an open syntaxin‐1 conformation that explains abundant experimental data and suggests an attractive hypothesis of how Munc13‐1 opens syntaxin‐1. These results indicate that a judicious selection of protein fragments can help AlphaFold to predict structures of kinetic intermediates in complex biomolecular processes.