Crystal Structure and Biophysical Properties of a Complex between the N-terminal SNARE Region of SNAP25 and Syntaxin 1a

Misura, Kira M. S.; Gonzalez, Lino C.; May, Andrew P.; Scheller, Richard H.; Weis, William I.

doi:10.1074/jbc.m106853200

Cited by 58 publications

(64 citation statements)

References 51 publications

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“…This suggests that in the presence of both SNAP-25 helices, a Q-SNARE complex containing two syntaxin molecules had formed. Interestingly, assembly in the presence of two independent helices of SNAP-25 was significantly slower and required much higher protein concentrations than with full-length SNAP-25, confirming previous results achieved by CD spectroscopy (24) and suggesting that both SNAP-25 helices cooperate for productive assembly.…”

Section: A Second Syntaxin Competes With Synaptobrevin For Binding Tosupporting

confidence: 76%

“…A, the SNARE motif of syntaxin (SyxH3) and the first helix of SNAP-25 (SN1) slowly form a stable assembly. This assembly exhibits a 2:2 stoichiometry and consists of an extended parallel four-helix bundle similar to that of the core SNARE complex (24). The SyxH3 2 SN1 2 complex probably represents an offpathway SNARE assembly, which forms via slow dimerization of a SyxH3-SN1 intermediate.…”

Section: Discussionmentioning

confidence: 99%

“…The molecular mass of the purified SyxH3-SN25⌬C complex determined by multiangle laser light scattering indicated a 2:2 stoichiometry (data not shown). An equivalent 2:2 complex has been described for the isolated first helix of SNAP-25 (SN1) and SyxH3 (24). Thus, in the absence of Syb, the much shortened second helix of the SN25⌬C construct may not suffice to prevent the competing but slow process of dimerization (for illustration, see Fig.…”

Section: A Second Syntaxin Competes With Synaptobrevin For Binding Tomentioning

confidence: 99%

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A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

Fasshauer

Margittai²

2004

Journal of Biological Chemistry

191

239

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The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca 2؉ -dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither Nnor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.Following a Ca 2ϩ influx into the synaptic terminal, neurotransmitter is rapidly released from synaptic vesicles that fuse with the plasma membrane. The synaptic vesicle protein synaptobrevin 2 (also referred to as vesicle-associated membrane protein 2, or VAMP 2) and the plasma membrane proteins syntaxin 1a and SNAP-25 1 play a central role during this process. They belong to the family of so-called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are essential for all vesicular trafficking steps. It is believed that SNAREs generally initiate the process of membrane fusion by the sequential formation of a stable heteromeric "trans" complex between the vesicular and the target membranes. This basic SNARE machinery is thought to be tightly controlled by a complex protein network (for a review, see Refs. 1-5).Syntaxin and synaptobrevin each contain a single SNARE complex-forming coiled-coil region, termed the SNARE motif, directly adjacent to their C-terminal transmembrane domain. Syntaxin 1a contains an additional regulatory N-terminal region, called the Habc domain. SNAP-25 is composed of two SNARE motifs connected by a long linker and is attached to the membrane by palmitoyl modifications in this region. Upon SNARE complex formation, major conformational changes occur, with mostly unstructured pr...

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Section: A Second Syntaxin Competes With Synaptobrevin For Binding Tosupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: A Second Syntaxin Competes With Synaptobrevin For Binding Tomentioning

confidence: 99%

See 1 more Smart Citation

A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

Fasshauer

Margittai²

2004

Journal of Biological Chemistry

191

239

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“…Complexes at greater than 200 kDa were prominent with CssY ( Figure 3A, lanes 2 and 5) but much less evident with CssYs ( Figure 3B, lanes 2 and 5). Both the apparent molecular weight and the strength of the bands suggest a complex of two CssYs and two syntaxins, analogous to the complex formed by two molecules of cytosolic SN1 and the H3 domain of syntaxin as determined by crystallography (Misura et al, 2001). The lack of SN2 probably promotes the formation of these quaternary complexes.…”

Section: Sds-resistant Complexes Formed By Fret Probes Syntaxin Andmentioning

confidence: 99%

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Wang¹,

Bittner²,

Axelrod

et al. 2008

MBoC

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We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDSresistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

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“…In addition to the well studied heterotrimeric neuronal and yeast plasma membrane SNARE complexes, other homo-and heteromeric interactions between individual SNAREs have been found and characterized (26)(27)(28). These alternate complexes are less stable than the heterotrimeric SNARE complex, but the relevance of these interactions is unknown.…”

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confidence: 99%

Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations

Weninger

Bowen

Chu

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

122

152

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Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h ؊1 . The existence of antiparallel configurations suggests a regulatory role of chaperones, such as N-ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly in vivo, and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments in vitro.A ll membrane fusion events in eukaryotes are thought to involve a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) (1, 2). The neuronal SNAREs syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25), and synaptobrevin are involved in the Ca 2ϩ -dependent fusion of synaptic vesicles with the presynaptic plasma membrane (3). Before synaptic vesicle docking, the individual SNAREs, syntaxin and SNAP-25, are primarily found on the plasma membrane, where they may exist as unfolded monomers, preformed binary complexes, or in complex with chaperones. At some point during docking and fusion of synaptic vesicles, heterotrimeric SNARE complexes form between syntaxin, SNAP-25, and the synaptic vesicle SNARE, synaptobrevin. SNARE complex formation is thought to occur first in trans with proteins on opposite membranes, and end with formation of a cis complex with proteins residing in the same membrane. Thus, SNARE complex crystal structures (4-7) probably represent the state of the SNARE complex at the endpoint of the fusion reaction (8).The inhibition of neurotransmitter release after the specific cleavage of any one of the SNAREs by clostridial neurotoxin proteases supports the fundamental role of the SNARE proteins in synaptic vesicle fusion (9, 10). However, whereas high concentrations of SNAREs have been shown to be sufficient to fuse synthetic liposomes in vitro (11), the molecular relationship between SNARE complex formation and Ca 2ϩ -triggered vesicle-membrane fusion in vivo remains unclear (12).Syntaxin, SNAP-25, and synaptobrevin have been the...

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Crystal Structure and Biophysical Properties of a Complex between the N-terminal SNARE Region of SNAP25 and Syntaxin 1a

Cited by 58 publications

References 51 publications

A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations

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