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

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

Zhang X1, Rebane AA1,2,3,4, Ma L1, Li F1,4, Jiao J1,2, Qu H1, Pincet F1,4,5, Rothman JE1,4, Zhang Y6.

Author information

1
Department of Cell Biology, Yale School of Medicine, New Haven, CT 06520.
2
Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06511.
3
Department of Physics, Yale University, New Haven, CT 06511.
4
Nanobiology Institute, Yale University, West Haven, CT 06477.
5
Laboratoire de Physique Statistique, École Normale Superieure, L'université de recherche Paris Sciences et Lettres, CNRS UMR 8550, Sorbonne Universités, Université Pierre-et-Marie-Curie University of Paris 06, Université Paris Diderot, 75005 Paris, France.
6
Department of Cell Biology, Yale School of Medicine, New Haven, CT 06520; yongli.zhang@yale.edu.

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

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