Structure and Dynamics of a Two-Helix SNARE Complex in Live Cells

Abstract

SNAREs are a superfamily of small membrane proteins catalyzing membrane fusion (1,2). They share as a common feature a conserved stretch of 60-70 amino acids, called the SNARE-motif. Most SNARE-motifs are membrane anchored by a C-terminal transmembrane region (TMR), and a few SNAREs possess two SNAREmotifs linked by a region containing a palmitoylated cysteine cluster for membrane attachment.Each fusion step requires a specific set of three to four SNAREs providing four SNARE-motifs. For instance, neuronal exocytosis is driven by formation of a complex between SNAP25 (containing two SNARE-motifs) and syntaxin 1A at the plasma membrane and vesicle associated synaptobrevin 2.The fusion reaction results in a ternary cis-SNARE complex in which the four SNARE-motifs adopt alpha-helical conformation, forming a parallel, twisted bundle stabilized by 16 layers of interaction (3). All layers are hydrophobic with the exception of a highly conserved central ionic layer to which each of the SNARE-motifs contributes either one out of three glutamines (consecutively named Q a , Q b and Q c ) or an arginine (R). According to their contributed amino acids, the SNAREs have been classified as Q a -(syntaxin), Q b -and Q c -(N-and C-terminal SNARE-motifs of SNAP25, respectively) and R-SNARE (synaptobrevin 2) (4). Retrospectively, the neuronal SNARE complex laid the ground for the structural Q/R-SNARE classification of all SNAREs discovered in the animal kingdom, fungi or plants.Although the post-fusion SNARE complex is well characterized, it is still debated by what sequence of interactions it assembles (5). Because Q-SNAREs form complexes but the R-SNARE-motif does not interact with any of the individual Q-SNAREs [for exception see e.g. (6)], it is generally believed that initially a Q-SNARE complex forms, which then functions as acceptor for the R-SNARE.However, in vitro soluble Q-SNARE-motifs (lacking their membrane anchor) do not form an intuitively expected stable Q a Q b Q c or Q x Q y complex, but have the intrinsic property to assemble into several four-helix bundles [e.g. (Q a ) 2 Q b Q c (7), (Q a ) 2 (Q b ) 2 (8) and (Q a ) 4 (8,9); for a SNAP23 (Q b ) 4 bundle see (10)].More recently, several groups accomplished to prevent the stabilization of Q a Q b Q c complexes into (Q a ) 2 Q b Q c complexes by different approaches. For instance, a Q a Q b Q c complex forms when SNAP25 is immobilized on sepharose beads by anti-SNAP25 antibodies (11), a C-terminal synaptobrevin fragment is used for stabilization (12), SNAP25 is available in large excess over syntaxin (12), or complexes are prepared in supported lipid bilayers containing syntaxin at a very low protein to lipid ratio (13).The latter study even allowed the characterization of single complexes and found several states. In addition to the Q a Q b Q c complex, other complexes were also observed with one of the SNAP25 SNARE-motifs dissociated (in principle representing a Q a Q b or Q a Q c complex). 394 www.traffic.dk

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

“…Studies using super-resolution imaging showed that syntaxin and SNAP25 form separate clusters with very little overlap, although they require each other for fusion (11)(12)(13). The clusters of syntaxin and SNAP25 are at equilibrium with free SNARE molecules, which are much more mobile (13,14).…”

Membrane Protein Clusters at Nanoscale Resolution: More Than Pretty Pictures

“…The preference for certain lipid phases can induce the formation of clusters. For example, GPI-anchored proteins have been found to associate with membrane rafts (33); also, the SNARE molecule SNAP25 (which lacks transmembrane domains) forms clusters that only depend on its palmitoyl chains, presumably integrating in the proper lipid phases, and not on protein-protein interactions (13,25). In a more complex description of membrane partitioning, the actin cytoskeleton may form fences in the plasma membrane, on which transmembrane proteins are anchored (16).…”

“…As an example, SNAP25 and syntaxin 1, proteins functioning in the vesicle fusion to the plasma membrane, are found "all over" the membrane, including areas where no vesicles could be found, so areas where they could not actually function (22). Also, although they are molecular partners with a common function, syntaxin 1 and SNAP25 are found in clusters that do not correlate particularly well (13).…”

STED imaging of green fluorescent nanodiamonds containing nitrogen-vacancy-nitrogen centers

“…It can be observed that the SNR is degraded because some part of the fluorescence signal is depleted but enough SNR can be maintained to isolate the single particles that were blurred in the confocal image. We demonstrated here superresolution imaging of gNDs inside fixed cells, however both the STED technique and the nanodiamond probes have been shown to be compatible with live cell imaging [5,8,24]. As the rNDs, gNDs, thanks to their high photostability, are useful probes when long acquisition with high resolution is required.…”

“…It results in breaking the diffraction limit of a fluorescence laser scanning microscope by depleting the fluorescence around the focus by stimulated emission. STED microscopy offers a resolution well below the diffraction limit [1,2], in the three dimensions [3], and it has been used to tackle many problems in biology [4,5]. However, the requirement of high power to obtain efficient depletion accelerates photobleaching and severely limits the observation time.…”