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.