Focused clamping of a single neuronal SNARE complex by complexin under high mechanical tension

Min Ju Shon,Haesoo Kim,Tae-Young Yoon

doi:10.1038/s41467-018-06122-3

Abstract

Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze synaptic vesicle fusion with presynaptic membranes through the formation of SNARE complexes. Complexin (Cpx) is the only presynaptic protein that tightly binds to SNAREs and regulates membrane fusion, but how it modulates the energy landscape of SNARE complex assembly, especially under mechanical tension on the complex, remains unclear. Here, using magnetic tweezers, we report how Cpx interacts with single SNARE complexes. The effects of Cpx manifest only under high mechanical tensions above 13 pN. Cpx stabilizes the central four-helix bundle of SNARE motifs and, at the same time, prevents the complete zippering of SNAREs by inhibiting linker-domain assembly. These results suggest that Cpx generates a focused clamp for the neuronal SNARE complex in a linker-open conformation. Our results provide a hint as to how Cpx cooperates with neuronal SNAREs to prime synaptic vesicles in preparation for synchronous neurotransmitter release.

Highlights

Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze synaptic vesicle fusion with presynaptic membranes through the formation of SNARE complexes
Because the DNA handles were attached to the C-terminal ends of synaptobrevin-2 and syntaxin-1A via two artificial cysteine residues[35], tension was generated from the C-terminus of the SNAREs, mimicking the force-loaded environment assumed for neuronal SNARE complexes during synaptic vesicle fusion[36] (Fig. 1a,b and Supplementary Fig. 2; transmembrane domains were truncated)
As we varied the force applied to the magnetic bead by moving the permanent magnets, we tracked the vertical position of the bead at 100 Hz to monitor the conformation of a single SNARE complex

Summary

Introduction

Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze synaptic vesicle fusion with presynaptic membranes through the formation of SNARE complexes. Cpx stabilizes the central four-helix bundle of SNARE motifs and, at the same time, prevents the complete zippering of SNAREs by inhibiting linker-domain assembly These results suggest that Cpx generates a focused clamp for the neuronal SNARE complex in a linker-open conformation. It has been presumed that the tension rapidly builds up when a synaptic vesicle approaches a presynaptic membrane because of the electrostatic repulsion, hydration barrier, and steric hindrance between the two fusing membranes[31,32] This tension in a SNARE complex was shown to significantly tilt the energy landscape that governs SNARE zippering processes[33,34]. While Cpx has been suggested to clamp partially zippered SNARE complexes and prevent membrane fusion[21,25,26,27,28], it has proven difficult to observe such an intermediate conformation of the neuronal SNARE complex directly, likely because of the transient nature of this state in the absence of applied tension

Methods

Results

Conclusion