Simulations Reveal Multiple Intermediates in the Unzipping Mechanism of Neuronal SNARE Complex

Abstract

The assembling of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein complex is a fundamental step in neuronal exocytosis, and it has been extensively studied in the last two decades. Yet, many details of this process remain inaccessible with the current experimental space and time resolution. Here, we study the zipping mechanism of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex computationally by using a coarse-grained model. We explore the different pathways available and analyze their dependence on the computational model employed. We reveal and characterize multiple intermediate states, in agreement with previous experimental findings. We use our model to analyze the influence of single-residue mutations on the thermodynamics of the folding process.

Figure 1

Schematic view of the SNARE complex. The four parallel helices of synaptobrevin (Sb), syntaxin (Sx), and SNAP25 form the core bundle of the complex. One arginine and three glutamine residues constitute the ionic layer, which divides the C-terminal domain (CTD; layers +1 to +8) from the N-terminal domain (NTD; layers −7 to −1). The linker domain (LD) of Sx and Sb and the linker loop of SNAP25 are also shown. Dashed colored lines schematically indicate the anchoring points on Sx, Sb, and SNAP25 to the lipid membranes. These are also the residues used in the definition of the pulling coordinates.

Figure 2

Free-energy landscape of SNARE unzipping as obtained from US simulations on the collective variable RC1, rescaled to model the effect of a constant pulling force F = 10.6 k_BT/nm. The free energy is plotted as a function of the distances between the C-terminal of the first helix of SNAP25 and the C-terminals of, respectively, Sb and Sx. The dashed black line indicates the opening of the ionic layer. Schematic representations of the structure of SNARE in the three main free-energy minima (folded, Sx unfolded, and Sb unfolded) are shown.

Figure 3

Intermediate states observed in the MD simulation of the SNARE complex mechanical unzipping. (A) A force-extension plot is shown. The force exerted by the virtual spring is shown as a function of the linear separation between the terminals during the pulling (blue) and unpulling (red) phases. Different states are marked by dashed orange lines. (B) A schematic view of the structure of the intermediates is shown. The LD and layers +5, +3, −2, and 0 (ionic) are outlined in the relevant states; nonreversible transitions from states 6–7 to 7–3 are represented by a single arrow.

Figure 4

Details of intermediate states 3, 4, and 5. (A) A schematic view of the structure of the three states is shown; residues V241 on Sx and A72 on Sb are highlighted in purple, and the ionic layer is highlighted in yellow. (B) The distribution of distance between residues V241 of Sx and A72 of Sb is shown. (C) The unstructured fraction for residues of Sb is shown in the different intermediates: left y axis represents the fraction of structures in which residue i is “unstructured,” i.e., has broken its native contacts (empty squares) or is not assuming an α-helix conformation (filled circles); the dashed yellow line indicates the position of the ionic layer; red triangles (right y axis) report the unstructured fraction as experimentally measured with EPR by Shin et al. (16).

Figure 5

Effects of modifications on the SBM potential energy on the SNARE free-energy profile. (A) Free energy as a function of the Sx-Sb terminal distance is shown rescaled with F = 13.4 k_BT/nm for noncharged SBM, F = 14.4 k_BT/nm for charged SBM; vertical dashed lines indicate the boundaries of the seven intermediate states (the exact value of each boundary is also reported next to each line). (B–D) Effects on the energetics of the intermediate states for mutations of different layers in the NTD (B), MD (C), and CTD (D) of the SNARE complex are shown.