Reluctance to membrane binding enables accessibility of the synaptobrevin SNARE motif for SNARE complex formation

Abstract

SNARE proteins play a critical role in intracellular membrane fusion by forming tight complexes that bring two membranes together and involve sequences called SNARE motifs. These motifs have a high tendency to form amphipathic coiled-coils that assemble into four-helix bundles, and often precede transmembrane regions. NMR studies in dodecylphosphocholine (DPC) micelles suggested that the N-terminal half of the SNARE motif from the neuronal SNARE synaptobrevin binds to membranes, which appeared to contradict previous biophysical studies of synaptobrevin in liposomes. NMR analyses of synaptobrevin reconstituted into nanodiscs and into liposomes now show that most of its SNARE motif, except for the basic C terminus, is highly flexible, exhibiting crosspeak patterns and transverse relaxation rates that are very similar to those observed in solution. Considering the proximity to the bilayer imposed by membrane anchoring, our data show that most of the synaptobrevin SNARE motif has a remarkable reluctance to bind membranes. This conclusion is further supported by NMR experiments showing that the soluble synaptobrevin SNARE motif does not bind to liposomes, even though it does bind to DPC micelles. These results show that nanodiscs provide a much better membrane model than DPC micelles in this system, and that most of the SNARE motif of membrane-anchored synaptobrevin is accessible for SNARE complex formation. We propose that the charge and hydrophobicity of SNARE motifs is optimized to enable formation of highly stable SNARE complexes while at the same time avoiding membrane binding, which could hinder SNARE complex assembly.neurotransmitter release | membrane proteins | membrane traffic T raffic at most eukaryotic membrane compartments is governed by members of conserved protein families that underlie a universal mechanism of intracellular membrane fusion (1). Particularly important among these proteins are the members of the SNARE family, which are characterized by sequences of about 60-70 amino acid residues that are known as SNARE motifs and often precede C-terminal transmembrane (TM) regions (2-5). Through these motifs, SNAREs from two apposed membranes form a tight four-helix bundle called the SNARE complex (6, 7), which brings the two membranes together and was proposed to provide the energy for membrane fusion (8). Although the exact fusion mechanism is still unclear (9), and fusion depends critically on other universal factors such as Sec1/Munc18 proteins (10-12) and sometimes on specialized proteins such as synaptotagmin-1 in the case of synaptic vesicle exocytosis (13-15), there is little doubt that the SNAREs play a central role in membrane fusion.Crystal structures of the SNARE four-helix bundle that represents the postfusion state have been determined without or with the adjacent TM regions (e.g., refs. 7 and 16), but detailed structural information on the isolated SNARE motifs attached to their adjacent TM regions is more limited. Early studies of synaptobrevin, syntaxin-1, and SNAP-25, the SNAREs that ...

A Plug Release Mechanism for Membrane Permeation by MLKL

“…Because nanodiscs of relatively small sizes (e.g. 10-12 nm) can be obtained by using appropriate fragments of apolipoprotein A1 (Denisov et al, 2004), they provide a powerful tool to analyze proteins in membrane environments by NMR spectroscopy using transversed relaxation optimized (TROSY)-based experiments (Shenkarev et al, 2009; Brewer et al, 2011; Hagn et al, 2013). Intriguingly, addition of nanodiscs containing 15% cardiolipin did not cause any significant changes in the positions and intensities of the 1 H- 15 N TROSY-HSQC cross-peaks of MLKL(2-154), whereas strong decreases in cross-peak intensities were observed upon addition of liposomes with the same phospholipid composition (compare black and red contours in the two-dimensional plots and the one-dimensional traces shown in Figures 2A,B).…”

Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states

“…Each S-v-nanodisc sample generated a narrow EPR spectrum reflecting unstructured conformation of VAMP-2 prior to complex formation with t-nanodisc [20] (Figure 3B, uncomplexed). Meanwhile, when the cis complex, which was prepared by premixing spin-labeled VAMP-2 with t-SNAREs, was reconstituted into nanodiscs, EPR spectra showed significant line-broadening due to formation of a stable four-helix bundle (Figure 3B, cis ).…”

Prevalent mechanism of membrane bridging by synaptotagmin-1

“…Nanodisc Preparation. Nanodiscs were prepared as previously described (47) with small modifications (48). Briefly, ApoA1 and lipids (DOPS:POPC 15:85) at an ApoA1:lipid ratio of 2:130 were mixed with 1% n-octyl-β-D-glucopyranoside and 1% sodium cholate.…”