EPR Analysis of SNARE Proteins: Transient States of the Neuronal Acceptor Complex

Neuronal fusion involves complex protein machinery that acts in a highly orchestrated manner when triggered by a neuronal action potential. This process involves three SNAREs, a highly conserved group of proteins that can interact to form a coiled-coil assembly called the SNARE complex, which is believed to provide the driving force for fusion of the neurotransmitter-arrying vesicle to the presynaptic space. Aside from SNAREs, other proteins, such as Munc13, synaptotagmin and Munc18 serve regulatory roles by acting on SNAREs. The study presented here used EPR spectroscopy to analyze the structure and interactions of syntaxin, SNAP-25, synaptobrevin (the three SNAREs), Munc18 and the MUN domain of Munc13. By performing a detailed CW spectral analysis, it was discovered that both syntaxin and synaptobrevin exist in conformational equilibria between ordered and disordered states. The structural equilibrium of syntaxin is also modulated by Munc18, likely controlling its interaction with other SNAREs. Pulsed EPR studies also revealed syntaxin as existing predominantly in a close tertiary structure, and that Munc18 further stabilizes this closed state. Munc18 also acts to suppress the syntaxin-SNAP25 binary complex, which is a dead-end configuration for SNAREmediated fusion. Taking all the EPR data together, the SNAREs are shown as engaging in complex interactions with Munc18 and MUN, which results in an intricate pathway of numerous protein structural states and transient intermolecular interactions.