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

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 k B T, where k B 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.t-SNARE complex | SNARE four-helix bundle | SNARE assembly | membrane fusion | optical tweezers S ynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate fast and calcium-triggered fusion of synaptic vesicles to presynaptic plasma membranes required for neurotransmission (1). They consist of VAMP2 (vesicle associated membrane protein 2, also called synaptobrevin 2) anchored on vesicles (v-SNARE) and syntaxin and SNAP-25 (synaptosome associated protein 25) located on target plasma membranes (t-SNAREs) (2). These SNAREs contain characteristic SNARE motifs of ∼60 amino acids (3) (Fig. 1A). Syntaxin and SNAP-25 can form a 1:1 t-SNARE complex (4-6). During membrane fusion, the t-and v-SNAREs join to form an extraordinarily stable four-helix bundle (3, 7-10). In the core of the bundle are 15 layers of hydrophobic amino acids and a central ionic layer containing three glutamines and one arginine. Whereas the zippering energy and kinetics between t-and v-SNAREs have recently been measured (8, 9), the structure, stability, and dynamics of the t-SNARE complex have not been well understood.The structure and dynamics of the t-SNARE complex are crucial for SNARE assembly and membrane fusion. Formation of the t-SNARE complex is likely an obligate intermediate before SNARE zippering (6,(11)(12)(13)(14). A preformed t-SNARE complex docks the vesicles to plasma membranes (15) and boosts the speed, strength, and accuracy of SNARE zippering (5, 9, 16). Furthermore, the t-SNARE complex is an important target for proteins that regulate SNAR...

Botulinum Toxins A and E Inflict Dynamic Destabilization on t-SNARE to Impair SNARE Assembly and Membrane Fusion

“…However, when bound to syntaxin 1A all positions except 203C exhibited a composite two component spectra with one broad component reflecting the structured species and another narrow component reflecting the unstructured species (Figure 1E and F). We did not observe much spectral change with 203C, consistent with the previous finding that the C-terminal end is frayed for the 1:1 complex (Zhang et al, 2016). …”

SNARE zippering requires activation by SNARE-like peptides in Sec1/Munc18 proteins

“…Since intracellular vesicle fusion is driven by the concerted action of SNAREs and SM proteins rather than by SNAREs alone (3), our findings provide a molecular explanation for the crucial roles of the +1 and +2 layers of the v-SNARE in vivo (11). The CTDs of t-SNAREs are partially unstructured (23,24), which may prevent proper SNARE zippering such that the +1 and +2 layers are dispensable. This hypothesis predicts that restructuring t-SNARE CTDs would achieve the same effect on SNARE zippering as the SM protein.…”

“…To test this possibility, we examined the regulatory activity of an engineered peptide corresponding to the CTD of the v-SNARE (Vc peptide) ( Fig. 3B), which binds t-SNARE CTDs and converts the latter into a helical configuration (23,25,26). We observed that Vc peptide markedly accelerated SNARE-mediated liposome fusion, and the Vc peptide-stimulated fusion reaction was highly sensitive to deletions in all CTD layers including the +1 and +2 layers ( Fig.…”

Single-molecule force spectroscopy of protein-membrane interactions

“…We treated the polypeptide tether using a Gaussian model for the polymer chain (Dill and Bromberg, 2010). Accordingly, the effective concentration c of the free end of the chain at a position r ¼ x; y; z ð Þ in the Cartesian coordinate or ; f; z ð Þ in the cylindrical coordinate is (Zhang et al, 2016a) c ¼ 1…”