Probing the Conformational Flexibility of the Munc18-1/Syntaxin-1A Complex

Abstract

Munc18-1, a protein of the Sec1/Munc18-1 (SM) protein family, is interacting with high affinity with Syntaxin-1a, a neuronal SNARE protein. Neuronal SNARE proteins constitute the core machinery that drives fusion of neurotransmitter-loaded synaptic vesicles with the plasma membrane. The tight interaction between Munc18-1 and Syntaxin-1a also controls neurotransmitter release. However, biochemically this interaction inhibits SNARE complex formation, because Syntaxin-1a is locked in a closed conformation by Munc18-1. In this conformation, the SNARE domain of Syntaxin-1a is bound inside the central cavity of Munc18-1. It remains unclear whether Syntaxin-1a has to leave the tight grip of Munc18-1 in order to adopt an open conformation that can then assemble into a SNARE complex or, whether, Munc18-1 remains bound, renders Syntaxin-1a open and by this facilitates SNARE complex formation. The latter scenario implies that the Munc18-1/Syntaxin-1a complex must be able to undergo conformational changes, a notion that is supported by Syntaxin-1a mutations that bypass the inhibitory activity of Munc18-1. Such changes might be triggered by additional factors such as Munc13 or induced by post-translational modifications. However, the sequence of conformational changes of the Munc18-1/Syntaxin-1a complex remains elusive so far. To shed more light on these putative conformational changes, we introduced mutations on regions of both proteins that could be involved in the switch. Then, we assessed whether these enabled bypassing of the Munc18-1 exerted inhibition of SNARE complex formation using biophysical and biochemical approaches. Results, so far, suggest that Syntaxin-1a opens up while in complex with Munc18-1.