Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins.

Keywords: SNARE assembly; energy landscape; membrane fusion; optical tweezers; protein folding; synaptic exocytosis.

Figure 1

SNARE domain structures, membrane fusion, and the experimental setup to study SNARE assembly. (A) Amino acid sequences and their domain structures of the SNARE motifs in synaptic syntaxin 1 (SX1), VAMP2, and SNAP‐25B (SN1 and SN2). The amino acids in the hydrophobic layers or the central ionic layer are highlighted in yellow. The amino acids colored red are mutated to test their effects on SNARE assembly. Syntaxin and VAMP2 are cross‐linked at their N‐termini, with one of the cross‐linking sites marked by a red rectangle, and pulled from their C‐termini. The extremely fast middle domain (MD) transition and its associated state 3 can only be resolved in some experiments and is often mixed with the CTD transition, as is shown in C. (B) SNARE proteins couple their folding and assembly to draw two membrane into proximity and force them to fuse. An assembled synaptic SNARE complex is depicted to bridge the plasma membrane and the vesicle membrane. (C) Experimental setup to pull a single cytoplasmic domain of the synaptic SNARE complex using optical tweezers (OTs). The assembled SNARE complex consists of an N‐terminal Habc domain in a antiparallel three‐helix bundle, the core four‐helix bundle domain, and a linker domain (LD) in a two‐stranded coiled coil. The four‐helix bundle contains an N‐terminal domain (NTD) and a C‐terminal domain (CTD) that are separated by a central ionic layer (“0” layer). Adapted from Ma et al.53

Figure 2

The SNARE complex zippers stepwise. (A) Force‐extension curves (FECs) obtained by first pulling and then relaxing single SNARE complexes in the absence (#1 and #2) and presence (#3) of SNAP‐25 in the solution. Regions of different states (red numbers, see B) are fit by the Marko‐Siggia formula (dashed red lines). Some transitions are marked: the reversible LD transition by a dashed oval, the reversible CTD transition by a solid oval, NTD unfolding by green arrows, t‐SNARE unfolding by cyan arrows, t‐SNARE refolding by the blue arrow, and full SNARE reassembly by red arrows. The inset shows the close‐up view of the CTD transition. (B) Derived SNARE assembly and disassembly states and pathway. The free energies of different states relative to the unzipped state (E) are indicated. Adapted from Gao et al. and Ma et al.48, 53

Figure 3

FECs of wild‐type (WT) and mutant SNARE complexes showing effects of layer mutations on SNARE assembly. The combined or mixed domain transitions are indicated by their domain names connected by “_” and “+,” respectively (also see legend for Figure 5). Adapted from Ma et al.53

Figure 4

Force‐dependent reversible folding and unfolding of the WT SNARE complex among four states. (A) Extension‐time trajectories (black) of a single SNARE complex at two indicated mean forces (F). The red trajectories are idealized state transitions derived from hidden‐Markov modeling (HMM). (B) Force‐dependent state populations (top, symbols) and transition rates (bottom) and their best model fits (solid or dashed curves). (C) Simplified energy landscapes66 of the wild‐type (WT) and mutant (L60A) SNARE complexes. The reaction coordinate is the number of the amino acid in VAMP2 located at the C‐terminal border of the zippered region that starts from the amino acid number 36 at the N‐terminal cross‐linking site [Fig. 1(A)]. From Ma et al.53

Figure 5

Position‐dependent effects of layer mutations on SNARE assembly. (A) Extension‐time trajectories of the WT and mutant SNARE complexes at the indicated constant mean forces (F). Domains that are involved in sequential folding and unfolding transitions are indicated by the domain names connected by “+” signs. However, some mutations cause two neighboring domains to cooperatively fold and unfold as single extended domains, which are indicated by two individual domains connected by “_” signs. (B) Probability‐density distributions of the extensions shown in A (symbols) and their best‐fits by sums of two to four Gaussian functions (curves). The red symbols and curves for L60A and L70A show the distributions of the extensions for NTD transitions (not shown) at high forces. From Ma et al.53

Figure 6

Effects of layer mutations on SNARE folding energy. Gray bars indicate the sum of the MD and NTD folding energies. Colored bars indicate energies of the combined domains denoted. All mutations are layer mutations in VAMP2, except the double mutations M71A and I192A in SNAP‐25 and T251I in syntaxin. Mutations highlighted in red correspond to impaired exocytosis. Reproduced from Ma et al.53

Figure 7

Different SNARE complexes share fast CTD folding, but exhibit different equilibrium forces and folding energies. (A) Extension‐time trajectories of two‐state CTD transitions in different SNARE complexes at different constant mean forces (F) near half unfolding probabilities (p). (B) CTD unfolding probability (symbol) as a function of force and its best model fit (solid curve). To compare folding dynamics of these SNARE complexes, we have used chimeric SNARE constructs in which the four SNARE motifs are joined into one polypeptide, which minimally interferes with CTD transitions and NTD unfolding. Adapted from Zorman et al.52

Figure 8

The synaptic t‐SNARE complex folds synchronously via a partially folded intermediate. (A) Extension‐time trajectories obtained by pulling the N‐terminal cross‐linked t‐SNARE complex from different C‐terminal sites (inset on the left). (B) Schematic diagrams of three t‐SNARE folding states (i–iii) and energies (E). Adapted from Zhang et al.55