Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

Proc Natl Acad Sci U S A. 2016 Dec 13;113(50):E8031-E8040. doi: 10.1073/pnas.1605748113. Epub 2016 Nov 28.

Abstract

Synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.

Keywords: SNARE assembly; SNARE four-helix bundle; membrane fusion; optical tweezers; t-SNARE complex.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Membrane Fusion
  • Mice
  • Microscopy, Atomic Force
  • Molecular Dynamics Simulation
  • Munc18 Proteins / chemistry
  • Munc18 Proteins / physiology
  • Optical Tweezers
  • Protein Conformation
  • Protein Domains
  • Protein Folding
  • Protein Stability
  • Qa-SNARE Proteins / chemistry
  • Qa-SNARE Proteins / physiology
  • SNARE Proteins / chemistry*
  • SNARE Proteins / genetics
  • SNARE Proteins / physiology
  • Synaptic Transmission / physiology
  • Synaptosomal-Associated Protein 25 / chemistry
  • Synaptosomal-Associated Protein 25 / physiology
  • Vesicle-Associated Membrane Protein 2 / chemistry
  • Vesicle-Associated Membrane Protein 2 / physiology

Substances

  • Munc18 Proteins
  • Qa-SNARE Proteins
  • SNARE Proteins
  • Snap25 protein, mouse
  • Synaptosomal-Associated Protein 25
  • Vesicle-Associated Membrane Protein 2
  • vesicle-associated membrane protein 2, mouse