SNARE motif

SNAREs is an acronym for soluble NSF acceptor protein receptors. They are a superfamily of small and mostly membrane-bound proteins that are distinguished by the presence of a conserved stretch of 60 amino acids referred to as a SNARE motif. With few exceptions, a single transmembrane domain is located adjacent to the SNARE motif at the C-terminal end. Many SNAREs possess in addition an independently folded N-terminal domain whose structures are more diverse. 

SNARE motifs spontaneously assemble into SNARE complexes. These consist of a bundle of four intertwined a-helices that are connected by a total of 16 layers of mostly hydrophobic amino acid side chains. In the middle of the bundle, there is a highly conserved and polar 0-layer consisting of three glutamine and one arginine residue. These residues are among the most conserved in the SNARE superfamily and led to a classification of SNAREs into Q- and R-SNAREs, respectively. Different fusion steps require different sets of SNAREs but some SNAREs can participate in different complexes, and some fusion steps involve several SNARE complexes that appear to operate in parallel and independently. 

Synaptic exocytosis involves three SNARE proteins the R-SNARE synaptobrevin/VAMP (isoforms 1 and 2) on the vesicle, and the Q-SNAREs syntaxin (isoforms 1 and 2) and SNAP-25 on the plasma membrane (Figure 4). Since SNAP-25 has two SNARE-motifs, synaptobrevin, syntaxin, and SNAP-25 together have four SNARE-motifs. Synaptobrevins and SNAP-25 are relatively simple SNARE proteins that are composed of little else besides SNARE motifs and membrane-attachment sequences (a transmembrane region for synaptobrevin, and a cysteine-rich palmitoylated sequence for SNAP-25). Syntaxins, in contrast, are complex proteins. The N-terminal two-thirds of syntaxins include a separate, autonomously folded domain (the so-called Habc-domain), while the C-terminal third is composed of a SNARE motif and transmembrane region just like synaptobrevin. 

Each intracellular fusion reaction exhibits characteristic properties, and involves a different combination of SM and SNARE proteins. The specificity of fusion reactions appears to be independent of SNARE proteins because SNARE complex formation is nonspecific as long as the Q/R-rule is not violated (i.e., the fact that SNARE complexes need to be formed by SNARE proteins containing R-, Qa-, Qb-, and Qc-SNARE motifs), and of SM proteins because SM proteins often function in... 

SNAREs comprise a superfamily of proteins that function in all membrane fusion steps of the secretory pathway within eukaryotic cells. They are small proteins that vary in structure and size (see Section 1.1), but share an evolutionary conserved stretch of 60-70 amino acids containing eight heptad repeats, which is termed SNARE motif (Brunger 2005). The number of different SNAREs varies between different organisms, ranging from 25 in yeast, 36 in mammals, to over 50 in plants. Each fusion step requires a specific set of four different SNARE motifs that is contributed by three or four different SNAREs, and each of the membranes destined to fuse contains at least one SNARE with a membrane anchor. 

Fig. 1 Structure of the neuronal SNAREs. Upper panel domain structure of the three neuronal SNARE proteins involved in synaptic vesicle fusion. Syntaxin 1A and SNAP-25 (contains two SNARE motifs) are associated with the presynaptic membrane, whereas synaptobrevin 2 is synaptic vesicle associated. The SNARE motifs form a stable complex (core complex) whose crystal structure has been analyzed (lower panel). In the complex, each of the SNARE motifs adopts an alpha-helical structure, and the four alpha-helices are aligned in parallel forming a twisted bundle (modified from Sutton et al. 1998). Stability of the complex is mediated by layers of interaction (—7 to +8) in which amino acids from each of the four alpha-helices participate (see text).

Synaptobrevin 2 is a small protein composed of 118 amino acids. It contains a SNARE motif with a short N-terminal proline-rich extension but lacks an independently folded N-terminal domain. Like syntaxin 1, the protein possesses a C-terminal transmembrane domain that is connected to the SNARE motif by a short linker (Figure 1). Synaptobrevin is palmitoylated at cysteine residues close to its transmembrane domain. Synaptobrevin 2 is highly expressed in neurons and neuroendocrine cells, but unlike syntaxin 1 it is also present in many non-neuronal tissues albeit at low levels. 

SNAP-25, a protein of 208 amino acids, deviates from the typical SNARE structure in that it has two SNARE motifs, joined by a flexible linker region, but lacks a transmembrane domain (Figure 1). The linker contains a cluster of four palmitoylated cysteine residues by which the protein is anchored at the plasma membrane. SNAP-25 can be phosphorylated at positions Thrl38 and Seri 87 by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), respectively. SNAP-25 represents a small subgroup of SNAREs with a similar structure, including SNAP-23, SNAP-29, and SNAP-47. In contrast to the neuron-specific SNAP-25 these SNAREs are ubiquitously expressed. 

As discussed above, fusion is driven by the assembly of SNAREs mediated by their SNARE motifs. Assembly is associated with a major release of energy, and consequently the SNAREs need to be refueled with energy by the generation of free SNAREs before they are reusable for another round of fusion. Thus, SNAREs undergo cyclic assembly and disassembly, and together the individual reactions involved are referred to as the conformational cycle of SNAREs (Figure 2). 

The final steps in fusion are only incompletely understood. For instance, it is controversial whether the non-bilayer transition states in fusion are initiated primarily by force, transmitted from the pull of the assembling SNARE motifs via the linkers onto the membrane, as suggested by mutagenesis of the linker domain. Alternatively, the function of the SNARE motifs may be confined to close apposition of the membranes, with the final steps being catalyzed by other factors such as a... 

Tomosyn is a soluble protein of 130 kDa with a C-terminal R-SNARE motif that is capable of replacing synaptobrevin in the neuronal SNARE complex. Most available data indicate that tomosyn negatively regulates exocytosis by competing with synaptobrevin in the formation of SNARE complexes (Brunger 2005), thereby leading to the inhibition of synaptic vesicle priming (McEwen et al. 2006). 

Rossetto O, Schiavo G, Montecucco C, Poulain B, Deloye F et al. (1994) Snare motif and neurotoxins. Nature 372 415-16... 

Solnble iV-ethylmaleimide-sensitive fnsion (NSF A -ethylmaleimide-sensitive factor) protein attachment protein receptors can be either R-SNAREs or Q-SNARES depending on sequence homologies SNARE motif Small nucleolar RNA Single nucleotide polymorphism Small nuclear RNA... 

SNARE motif and neurotoxin recognition. In Nature 372 415-6 Rossetto O, Gorza L, Schiavo G, Schiavo N, Scheller RH, Montecucco C (1996) ... 

The structure and interfacial association of the full-length vesicle SNARE, synaptobrevin (I), were compared in 4 different lipid environments using NMR and ESR spectroscopy. In micelles, segments of the SNARE motif were helical and associated with the interface. However, the fraction of helix and interfacial association decreased as I was moved from micelle to bicelle to bilayer environments, indicating that the tendency toward interfacial association was sensitive to membrane curvature. In bilayers, the SNARE motif of I transiently associated with the lipid interface, and regions that were helical in micelles were in conformational and environmental exchange in bicelles and bilayers. ... 

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