The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion.

Bin Lu

doi:10.7717/peerj.1065

Abstract

Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

Highlights

The assembly of target SNAREs (t-SNAREs), syntaxin 1A and SNAP-25, on the plasma membrane with the vesicular SNARE (v-SNARE), synaptobrevin 2 (VAMP2), is crucial for Ca2+-triggered regulated exocytosis (Poirier et al, 1998; Sollner et al, 1993; Sutton et al, 1998; Weber et al, 1998)
The conformation of the C-terminus of SNAP-25 SN1 and SN2 To compare the SN1 and SN2 conformations in the t-SNAREs binary complex, we employed site-directed spin labeling (SDSL) and continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy and prepared three cysteine mutants located in the C-terminus of SN1 and SN2, respectively (Fig. 1A): C63, 70 and 77 of SNAP-25 SN1, and C184, 191 and 198 of SNAP-25 SN2
Neurotransmitters release requires the complete assembly of three SNAREs, namely syntaxin 1A and SNAP-25 on the target plasma membrane and synaptobrevin 2 (VAMP2)

Summary

Introduction

The assembly of target SNAREs (t-SNAREs), syntaxin 1A and SNAP-25, on the plasma membrane with the vesicular SNARE (v-SNARE), synaptobrevin 2 (VAMP2), is crucial for Ca2+-triggered regulated exocytosis (Poirier et al, 1998; Sollner et al, 1993; Sutton et al, 1998; Weber et al, 1998). One study suggests that the three SNARE proteins do not assemble into any intermediate complex before the activation of exocytosis (Lang et al, 2002). Other studies show that t-SNARE proteins may form the binary complex first and engage with VAMP2 to form the ternary complex (An & Almers, 2004; Hu et al, 2002; Sorensen et al, 2006). Syntaxin 1A may interact with SNAP-25 at a 1:1 or 2:1 molar ratio

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Results

Conclusion