Munc18-1 induces conformational changes of syntaxin-1 in multiple intermediates for SNARE assembly

Sanghwa Lee,Jaeil Shin,Cherlhyun Jeong,Younghun Jung,Dae-Hyuk Kweon,Jonghyeok Shin,Yeon-Kyun Shin,Heyjin Son

doi:10.1038/s41598-020-68476-3

Abstract

In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex.

Highlights

In neuronal exocytosis, sensitive factor attachment protein receptor (SNARE) assembly into a stable four-helix bundle drives membrane fusion
The kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/ Munc[] complex is less stable than that in the closed syntaxin-1/Munc[] complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex
As an initial step toward the understanding of the regulation by Munc[], we investigated if Munc[] forms a ternary complex with SNAP-25 and syntaxin-1 and induces the closed conformation of syntaxin-1 to possibly prevent the syntaxin-1 from the unregulated binding of the vesicle SNARE (v-SNARE) synaptobrevin

Summary

Introduction

SNARE assembly into a stable four-helix bundle drives membrane fusion. We found that single Munc[] proteins induce the closed conformation of syntaxin-1 in the free syntaxin-1 and in the t-SNARE (syntaxin-1/SNAP-25) complex These results implicate that Munc[] may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc[]. It is known that Munc[] stimulates membrane fusion when it binds to a fully assembled SNARE complex[16,17,18] Despite such major advances, many important questions concerning the mechanisms underlying the precise regulation of SNARE complex formation by the Munc[] and Munc[] still remain unanswered. The structural and dynamic features of intermediates remain elusive

Methods

Results

Conclusion