Snaps Snares Proteins

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 Snaps Snares Proteins Background

First introduced in 1993, the term “SNARE”, stands for Soluble N–ethylmaleimide sensitive factor (NSF) attachment protein receptor. SNAREs are a large superfamily of small membrane proteins consisting of more than 60 members in yeast and mammalian cells. SNARE proteins are thought to be essential for membrane fusions during all the trafficking steps of eukaryotic secretory pathways. Identification of neuronal SNAREs that mediate synaptic vesicle fusion during neurotransmission suggests a universal SNARE-mediated mechanism of vesicle fusion common to both constitutive and regulated membrane trafficking.

Structurally, all SNARE proteins share an evolutionarily conserved domain of about 60 amino acids arranged in heptad repeats, of which the secondary structure is a coiled-coil of α-helix. This structural feature known as the SNARE motif is functionally important because it mediates the association of SNAREs into core complexes. In addition, most SNAREs have a C-terminal transmembrane (TM) domain that is linked to the SNARE motif by a short linker. Positioned N-terminal to the SNARE motif, many SNAREs have independently folded domains that mediate additional protein-protein interactions. A subset of SNAREs lacks the transmembrane domain, and most of these achieve membrane anchorage through hydrophobic post-translational modifications such as palmitoylation.

Different sets of SNAREs are found on two opposing membranes that are destined to fuse. Based on the membrane type they associate with, SNAREs are classified as vesicle associated, v-SNAREs, and target membrane associated, t-SNAREs. However, this terminology is not applicable to homotypic fusion events. Also, all SNAREs are evolutionarily related to each other and localization of trafficking vesicle or target membrane does not always correlate with structurally identified SNARE subfamilies. Because crystal structures show that a central residue in the SNARE motif is either a glutamine or an arginine, a preferred classification categorizes SNAREs as Q- and R-SNAREs based on the type of this central amino acid.

To date, the best characterized SNARE proteins are the ones involved in synaptic exocytosis. Neuronal SNAREs include synaptobrevin (SYB) which is also known as vesicle-associated membrane protein or VAMP, syntaxin (SYX), and synaptosomal-associated protein of 25 kDa (SNAP-25). SYB or VAMP was initially discovered as an integral membrane protein of 18kDa present in small synaptic vesicles in neurons. In human, 2 highly conserved homologous SYB 1 and 2, are encoded by separate genes SYB1 and SYB2, respectively. SYX (35kDa) and SNAP-25 (25kDa) were initially identified as neuronal proteins that are concentrated on the plasma membrane at synaptic sites. Both proteins were implicated in synaptic vesicle priming at presynaptic active zones. SYB was classified as a v-SNARE due to its vesicular anchoring, while SYX and SNAP-25 were categorized as t-SNAREs because of their plasma membrane localizations.

SYB has a single SNARE motif, which makes up most of the cytosolic portion of this protein and links to its C-terminal membrane anchor. SNAP-25 contains two different SNARE motifs located at its N- and C-termini. These two coiled-coil domains are connected by a flexible linker that contains 4 palmitoylated cysteine residues to mediate membrane anchorage. SYX has a single SNARE motif, the cytosolic H3 domain, flanked by a C-terminal transmembrane domain and an N-terminal cytosolic fragment containing three coiled-coil domains, Ha, Hb and Hc. These α-helices are structurally similar to SNARE motifs and can interact with the H3 domain to fold SYX into a closed conformation unable to bind other SNAREs. Switching between the open and closed conformations of SYX requires the function of additional regulatory factors, including nSec-1, Rab and Rab effectors.

When associated in a 1:1:1 stoichiometry, SYB, SYX and SNAP-25 form a SNARE complex also known as the core complex. Assembly of the ternary core complex is through interactions of SNARE motifs of the three SNAREs. Specifically, the core complex is a four helical bundle with SYB contributing its cytosolic coiled-coil domain, SYX contributing its H3 domain and SNAP-25 contributing both C- and N- terminal coiled-coils. The coiled-coil domains of SNAREs associate in a parallel alignment. That is, the N-termini are adjacent to one another with the C-termini adjacent to one another on the opposite end. Such alignment brings the umtransmembrane domains of SYB and SYX close together and thus puts the synaptic vesicle membrane into close opposition to the plasma membrane.

Structure of the SNARE core complex I

Figure 1 Structure of the SNARE core complex I.

Full assembly of the core complex may go through several conformational intermediates. Binding of SYX and SNAP-25 to form a binary receptor complex on the plasma membrane awaiting the addition of SYB may be the first step of core complex nucleation. During vesicle priming, a loose ternary SNARE complex is formed in trans between the membranes. Frequently referred to as the trans-SNARE complex, this conformation is formed through partial association of the SNARE motifs at the N-terminal ends, while the membrane proximal C-termini of SNARE motifs and the transmembrane domains of SYX and SYB remain disassociated. Complete core complex nucleation is through a rapid N- to C-terminal zippering of SNARE motifs triggered by a fusion signal.

Fully assembled core complexes, corresponding to cis-SNARE complexes formed after complete synaptic vesicle fusion, are extremely energetically favorable and exhibit extraordinary stability. Specifically, the core complex resists denaturation by SDS and exhibits great heat tolerance with a melting temperature of greater than 90 °C. In monomeric or partially assembled states, the 3 neuronal SNAREs are substrates of clostridial neurotoxins. Cleavage of SYB by tetanus toxin and botulinum toxin types B, D, F and G, SNAP-25 by botulinum toxins A, C and E or SYX by botulinum toxin C produces rapid inhibition of neurotransmission. However, full assembly of core complexes results in resistance to prototypical cleavage by these toxins, because cleavage sites are buried within the intertwined bundle of helices.

The above biochemical, structural and functional data support the following model of SNARE-mediated synaptic vesicle membrane fusion. Membrane fusion is initiated via assembly of the SNARE core complex. The SYX H3 domain is liberated from association with the Habc domain and binds to SNAP-25 to form a binary receptor complex to which SYB is added. During priming, the ternary SNARE complex is partially assembled via association of the N-termini of SNARE motifs, and undergoes rapid zippering towards the C-termini upon Ca2+ triggering. Helical extension proceeds beyond the core complex into the linker regions between SNARE motifs and transmembrane domains, which brings the vesicle and plasma membrane into contact and disrupts the lipid bilayer structure to initiate membrane merger. The repulsion force between the phospholipids is likely overcome by the free energy released during formation of this extremely stable coiled bundle. After membrane fusion, resultant cis-SNARE complexes are disassembled to release free SNAREs to participate in future rounds of vesicle priming and fusion. Disassembly of this stable protein complex requires the ATPase NSF (N–ethylmaleimide sensitive factor) and its cofactor SNAP (soluble NSF attachment protein).