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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.

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Department of Cell Biology, Yale School of Medicine, New Haven, CT 06520.
Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06511.
Department of Physics, Yale University, New Haven, CT 06511.
Nanobiology Institute, Yale University, West Haven, CT 06477.
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.
Department of Cell Biology, Yale School of Medicine, New Haven, CT 06520;


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.


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

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