From the Division of Hemostasis and Thrombosis, Beth
Israel Deaconess Medical Center, Boston, Massachusetts 02115, the
Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, Massachusetts 02115, and the
§ Department of Cancer Biology, Dana-Farber Cancer
Institute, Boston, Massachusetts 02115
Received for publication, October 13, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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SNARE proteins mediate intracellular membrane
fusion by forming a coiled-coil complex to merge opposing membranes. A
"fusion-active" neuronal SNARE complex is a parallel four-helix
bundle containing two coiled-coil domains from SNAP-25 and one
coiled-coil domain each from syntaxin-1a and VAMP-2. "Prefusion"
assembly intermediate complexes can also form from these SNAREs.
We studied the N-terminal coiled-coil domain of SNAP-23
(SNAP-23N), a non-neuronal homologue of SNAP-25, and its interaction
with other coiled-coil domains. SNAP-23N can assemble spontaneously
with the coiled-coil domains from SNAP-23C, syntaxin-4, and VAMP-3 to
form a heterotetrameric complex. Unexpectedly, pure SNAP-23N
crystallizes as a coiled-coil homotetrameric complex. The four helices
have a parallel orientation and are symmetrical about the long axis.
The complex is stabilized through the interaction of conserved
hydrophobic residues comprising the a and d
positions of the coiled-coil heptad repeats. In addition, a central,
highly conserved glutamine residue (Gln-48) is buried within the
interface by hydrogen bonding between glutamine side chains derived
from adjacent subunits and to solvent molecules. A comparison of the
SNAP-23N structure to other SNARE complex structures reveals how a
simple coiled-coil motif can form diverse SNARE complexes.
Endosomal trafficking and vesicle secretion in eukaryotic cells
requires fusion of membrane compartments. Synaptic vesicle secretion in
neurons has served as a paradigm for understanding the molecular basis
for membrane fusion (1). In general, a v-SNARE1 called VAMP
(synaptobrevin) is associated with the vesicle membrane and two
t-SNAREs called SNAP-25 and syntaxin are associated with the plasma or
target membrane. According to the SNARE hypothesis, specific pairing
between v- and t-SNAREs brings together two membranes for fusion (2).
Following fusion, NSF and SNAPs dissociate the SNARE complex in an
ATP-dependent reaction (3).
SNARE proteins contain coiled-coil regions that mediate complex
formation (4). VAMP and syntaxin proteins each have a single SNARE-interacting coiled-coil domain followed by a C-terminal transmembrane domain, whereas SNAP-25 proteins have N- and C-terminal coiled-coil domains separated by a loop that attaches to membranes via
palmitoylated cysteines (5). The coiled-coil sequences are highly
conserved among SNARE families (4). The homologous regions are made up
of ~8 heptad repeats in which the hydrophobic residues in the
a and d positions of each repeat align in 16 layers (4). The central "0" layer is uniquely polar and consists of a glutamine in syntaxin and the SNAP-25 N- and C-terminal coiled-coil domains and an arginine in the VAMP coiled-coil domain. The structure of a core SNARE complex from neurons revealed a parallel four-helix bundle composed of two coiled-coil domains from SNAP-25, and one coiled-coil domain each from syntaxin-1a and VAMP-2 (6, 7). The
parallel orientation of SNARE proteins attached to opposite membranes
suggests a "zipper-like" mechanism for membrane fusion (7). A
distantly related endosomal SNARE complex has a very similar structure,
suggesting a common fusion mechanism mediated by neuronal, endosomal,
and possibly other SNARE complexes (8).
SNARE isoforms SNAP-23, syntaxin-4, and VAMP-3 (Cellubrevin) are
present in many non-neuronal cell types (9-12). Foster et al. (13) found that soluble forms of SNAP-23, syntaxin-4, and VAMP-2 (present in neurons and other cells) formed binary complexes but
not a native-like ternary complex. In contrast, Yang et al. (14) found that the coiled-coil domains of these proteins could form a
heterotetrameric complex. However, the stability of this non-neuronal
SNARE complex was decreased relative to a neuronal one composed of
SNAP-25, syntaxin-1a, and VAMP-2. These studies suggest that this group
of non-neuronal SNARE proteins may function differently than the
neuronal ones.
The structures of non-neuronal SNAREs and their complexes are not well
characterized. We used a structural approach to understand the function
of non-neuronal SNARE proteins involved in membrane fusion.
Interestingly, we find that SNAP-23N (Fig.
1) can form a parallel four-helix bundle
in the absence of other SNARE proteins. The 2.3-Å crystal structure of
SNAP-23N reveals important similarities and differences with other
SNARE complex structures. Comparison of these structures extends our
understanding of how SNARE proteins assemble and function.
Protein Expression and Purification--
PCR DNA fragments
encoding amino acids 19-71 of rat VAMP-3 (100% identical to human),
210-262 of rat syntaxin-4 (94% identical to human), and 23-76 and
156-208 of human SNAP-23 were cloned into the BamHI and
EcoRI sites of a pET vector (Novagen) that expresses
glutathione S-transferase (GST) fusion proteins containing a
thrombin protease site. The expression plasmid introduces a vector-derived Gly-Ser at the N terminus of each peptide that remains
after thrombin cleavage. The constructs were transformed individually
into BL21 (DE3) Escherichia coli cells. To prepare selenomethionine-substituted SNAP-23N (SeMet-SNAP-23N), the
SNAP-23N-expressing plasmid was transformed into B834 (DE3) methionine
auxotroph E. coli cells and grown in M9 minimal
medium containing a complete complement of amino acids except
for methionine substituted by selenomethionine (Sigma). Bacteria were
grown in 50 µg/ml ampicillin, and expression was induced with
isopropyl-1-thio- SNARE Binding Assay--
Approximately equimolar amounts (20 µM) of VAMP-3, syntaxin-4, SNAP-23N, and SNAP-23C
coiled-coil peptides were mixed together in the indicated combinations
and incubated at 4 °C for 2 h. The binding buffer contained 50 mM Tris-HCl, pH 7.5, and 50 mM NaCl. Aliquots
of each binding reaction were analyzed by non-denaturing electrophoresis on 10% polyacrylamide gels.
Crystallization, X-ray Diffraction Data Collection, and
Phasing--
Underivatized SNAP-23N was crystallized by the hanging
drop vapor diffusion method at 23 °C. SNAP-23N was crystallized at a
concentration of 1-4 mg/ml by combining 1 µl of purified protein in
50 mM Tris-HCl, pH 7.5, 100 mM NaCl with 1 µl
of reservoir solution containing 0.1 M Hepes, pH 7.5, 0.2 M CaCl2, and 28% polyethylene glycol (PEG)
400. Typically, crystals were observed within 24 h and grew to
maximal size by 48-72 h. SeMet-SNAP-23N was crystallized using the
same buffer conditions, but at 4 °C.
Diffraction data were collected by flash-freezing crystals from the
mother liquor in the cryostream at 100 K. Native diffraction data were
recorded on a 300 mm MarResearch image plate detector using a rotating
anode source with mirror optics (OsmicTM). Multiwavelength
anomalous diffraction (MAD) data were recorded on a Brandeis
four-module CCD detector at beamline X-12C of the National Synchrotron
Light Source of Brookhaven National Laboratories. Data were processed
and scaled using DENZO/SCALEPACK (15) for home data and HKL2000 (15)
for MAD data. Native crystals diffracted x-rays on the home detector to
2.5-Å resolution and SeMet crystals diffracted at the synchrotron
source to 2.3 Å resolution. Crystallographic data are summarized in
Table I.
Structure Determination--
Molecular replacement with COMO
(16) using the coordinates for rat SNAP-25N (7) as a search model
yielded clear rotation and translation solutions for the peptide. The
resulting 2Fo Non-neuronal SNARE peptides from SNAP-23, syntaxin-4, and VAMP-3
were expressed recombinantly in bacteria and purified as described
under "Experimental Procedures." The N- and C-terminal boundaries
were chosen based on homology to the coiled-coil regions in the
neuronal SNARE complex (4). Each peptide is a 55-mer (including the
vector-derived N-terminal Gly-Ser) that correponds to 7.5 heptad
repeats. All peptides except the SNAP-23C peptide were soluble in the
millimolar range in aqueous solution; SNAP-23C has a pI of 9.39, which
may contribute to its modest solubility. A binding assay using native
polyacrylamide gel electrophoresis was performed to determine whether
the SNARE peptides could interact with each other. Mixing equimolar
amounts of all four peptides yielded a new complex band not observed
with any of the three-way combinations of peptides (Fig.
2A). In another binding
experiment, GST-SNAP-23C attached to glutathione beads was mixed with
the other three SNARE peptides, and a pure peptide complex was eluted by thrombin cleavage. This purified complex had similar electrophoretic mobility on a native gel (Fig. 2B) and contained the
coiled-coil domains of four SNARE peptides as verified by MALDI-TOF
mass spectroscopy. We conclude that the coiled-coil domains from
SNAP-23, syntaxin-4, and VAMP-3 can form a heterotetrameric complex. To
date, we have been unable to obtain crystals of this heterotetramer, in
part due to the poor solubility of SNAP-23C, which has prevented
preparation of large quantities of the complex. However, we did observe
small crystals in an initial screen in which equimolar amounts of
SNAP-23N, SNAP-23C, syntaxin-4, and VAMP-3 were combined. Surprisingly, analysis of these crystals by mass spectroscopy demonstrated that they
contained only SNAP-23N. It is likely that rapid precipitation of
SNAP-23C, and possibly other SNARE peptides, in this experiment prevented formation of the expected heterotetramer. Subsequently, we
determined the SNAP-23N structure to provide insight into its quaternary structure and potential function. The human SNAP-23N sequence is shown aligned with homologous sequences in Fig. 1.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Helical wheel diagram of the SNAP-23N
homotetramer showing residues 24-76. View is from the N
terminus, and residues in the first two helical turns are
circled. Heptad positions are labeled a through
g. Note that residues that make intermolecular contacts are
located in heptad positions a and d and make 16 intermolecular planes or "layers." A central highly conserved
glutamine residue is highlighted in blue, and the two
methionines derivatized with selenium for crystallographic phasing are
highlighted in red. Below, alignment of SNAP-23N and
SNAP-25N sequences is shown. Conserved heptad repeat a and
d residues are highlighted in yellow, and the
central conserved glutamine is highlighted in blue. The
aligned sequences are: h, Homo sapiens;
r, Rattus norvegicus; m, Mus
musculus; x, Xenopus laevis;
s, Sachharomyces cervesiae (SNAP-25 yeast
homologue called sec9). The top figure was adapted from
Harbury et al. (27).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 3 h at
37 °C beginning at OD600 = 0.5-0.6. Bacteria were lysed by sonication in 50 mM Tris-HCl, pH 7.5, 100 mM
NaCl, and one complete protease inhibitor tablet (Roche Molecular
Biochemicals), and the peptides in the supernatant fraction were
isolated over glutathione-Sepharose 4B resin (Amersham Biosciences).
The peptides were eluted with thrombin protease and further purified
over a Superdex 75 gel filtration column equilibrated in 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl using an
ÄKTApurifier (Amersham Biosciences). SNAP-23C is poorly soluble
after concentration, but an adequate amount for the binding assay (see
"Results") could be achieved by directly using the SNAP-23C eluate
from purifed GST-SNAP-23C attached to glutathione beads. Peptides were
concentrated over YM-3 microcon membranes (Millipore), and their final
concentrations were determined using the Coomassie plus protein assay
reagent (Pierce) with bovine serum albumin as a standard. Purity was
determined by SDS-PAGE and MALDI-TOF mass spectroscopy.
Crystallographic data
Fc electron
density map was readily interpretable for the N-terminal region of
SNAP-23N, but weak density for the more C-terminal region of the
structure complicated rebuilding and refinement. Therefore, we recorded
a three-wavelength MAD data set with SeMet-substituted peptide. An
electron density map generated from the MAD data confirmed the
molecular replacement solution and revealed clear density for most of
the peptide sequence. However, density for the C-terminal 8 residues
was relatively weak. The selenium positions of the 2 SeMet residues
were located and refined with SOLVE (17) using data from 20 to 3 Å.
Manual rebuilding with O (18) and refinement with CNS (19) to the
higher resolution SeMet data produced a final model with an
R value of 27.3% and a free R value of 31.6%.
The higher than expected final R factor may be a consequence
of the relatively weak density and high thermal parameters for the
C-terminal 8 residues in the structure. Similar R factors
were reported for a related SNARE complex for similar reasons (20). The
final model contains residues 23-76 of SNAP-23N and 43 solvent molecules.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
SNARE peptide binding assay.
A, individual SNARE peptides (first four lanes)
and mixtures of SNARE peptides (last six lanes) are shown on
a non-denaturing polyacrylamide gel. The last two lanes are
a duplicate experiment of all four peptides mixed together. The
SNAP-23C peptide (asterisk) migrates poorly due to its high
pI (9.39), and the weaker band beneath it is contaminating GST
(open arrow). Coomassie-stained protein bands other than the
individual forms represent heterodimeric and heterotrimeric complexes
of SNARE peptides as reported by others (22). Note that the last
two lanes contain a new, slowly migrating "diffuse" band
(closed arrow) that is not present in lanes containing
three-way combinations of peptides. These bands represent the putative
SNARE peptide heterotetramer. B, purification of the SNARE
peptide heterotetramer (lane 2) from a mixture of purified
SNAP-23N, SNAP-23C, syntaxin-4, and GST-SNAP-23C (lane 1).
The composition of this pure band containing all four peptides
was determined by mass spectroscopy (see "Results" for
details).
Purified SNAP-23N crystallized in the presence of divalent metal ions (e.g. CaCl2 or MgCl2). The native crystals are rectangular prisms with approximate dimensions of 0.08 × 0.02 × 0.02 mm3, whereas the SeMet crystals are square plates with approximate dimensions of 0.13 × 0.13 × 0.01 mm3. Both crystal forms have the same tetragonal space group with one copy of SNAP-23N in the asymmetric unit. Symmetry constraints resulting from the 4-fold symmetry of the space group suggested that SNAP-23N molecules were organized as a parallel homotetrameric complex. Structure determination confirmed this prediction (see below).
Each SNAP-23N molecule is an extended amphipathic helix with 14 helical turns. The left-handed superhelical twist of the complex
results in a ~13° bend in each helix (Fig. 4A). The
electron density map shows excellent main chain connectivity along its entire length from Ser-23 to Leu-76 (Fig.
3). However, the side chain conformations
of surface-exposed residues between Lys-64 and Leu-81 are less well
defined. Four molecules of SNAP-23N produce a parallel four-helix
bundle with overall dimensions of ~80 × 24 × 24 Å3 (Fig. 4, A and
B). Each peptide in the complex has the same conformation, and thus, the tetramer has exact 4-fold symmetry as required by the
tetragonal crystal lattice. The SNAP-23N peptide is acidic (pI = 4.70), and the complex displays a mostly electronegative solvent-exposed surface; in contrast, the complex interface is mostly
neutral (Fig. 4C).
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The homotetrameric complex is held together primarily through
hydrophobic interactions (Fig.
5A). Hydrophobic residues
occupy the a and d positions of the coiled-coil
heptad repeats, and their side chains face the interior of the complex
(Fig. 1). The only exception is Gln-48 whose polar side chain is also
buried in the interface (Fig. 5B). Thus, planes of buried
residues occur at regular intervals along the chain and create 16 layers of intermolecular contacts. Gln-48 is located in the central or
0 layer as in other SNARE complexes. Our structure shows that
the polar groups of the Gln-48 side chains point toward the center of
the complex and are only 2.5 Å apart from one another (Fig.
5B). Although it is a deviation from the strict
crystallographic symmetry, we have modeled the four Gln-48 residues
with alternating side chain rotamers as it allows chemically reasonable
hydrogen bonding in the tetramer. In addition, a water molecule
hydrogen bonds to the free polar group not coordinated by a Gln-48 side
chain in an adjacent molecule (Fig. 5B).
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Although Ca2+ is required for secretion in a variety of
cell types (21) and divalent cations were required for crystallization, Ca2+ ions were not observed in the electron density map in
the crystal structure. Thus, Ca2+ ions may have a
nonspecific stabilizing effect on the SNARE complex structure.
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DISCUSSION |
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We found that SNAP-23N can associate with other non-neuronal SNARE coiled-coil domains to form a minimum heterotetrameric complex. This result is in agreement with the findings of Yang et al. (14) who used coiled-coil domains from SNAP-23, syntaxin-4, and VAMP-2 (98% identical to VAMP-3) but conflicts with the work of Foster et al. (13) who could not detect a ternary complex using GST-fused cytoplasmic regions of the same proteins. It is possible that the assay conditions of the latter study were not suitable for creating a ternary complex.
Work by others (22, 23) indicated that the neuronal SNAP-23N homologue, SNAP-25N, is mostly unstructured in solution. SNAP-23N and SNAP-25N are 72% identical and so might be expected to behave similarly. On the other hand, heterotetrameric coiled-coil complexes containing SNAP-25 have a higher stability compared with those containing SNAP-23 (14); perhaps the driving force for SNAP-23N self-association destabilizes its interaction with other SNARE coiled-coil domains.
Comparison of the SNAP-23N structure to other SNARE complexes reveals
important similarities and differences. Like the SNAP-23N homotetramer,
the neuronal heterotetramer and complexes between the SNAP-25
coiled-coil domains with the syntaxin-1a coiled-coil domain are
arranged as parallel four-helix bundles (7, 23-25). In contrast, a
syntaxin-1a homotetramer contains two pairs of helices that run
antiparallel to one another (26). The neuronal heterotetrameric complex
has a much larger left-handed helical twist compared with the SNAP-23N
homotetramer, because each chain in the heterotetramer has a slightly
different conformation. Despite these differences the SNAP-23N
homotetramer and neuronal heterotetramer appear remarkably similar
(Fig. 6B). In fact,
superimposition of the SNAP-23N and SNAP-25N chains reveals a
backbone root mean square deviation of only 1.1 Å in which greatest
variability occurs over a small region at the C terminus (Fig.
6A). This structural difference may stem from an inherent
difference between SNAP-23 and SNAP-25, the conformational lability of
the SNAP-23N C-terminal region, or the fact that SNAP-23N in a
homotetrameric complex is being compared with SNAP-25N in a
heterotetrameric complex.
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Hydrophobic intermolecular contacts are found in all SNARE complex
structures. Interestingly, the packing of hydrophobic a and
d residues (Fig. 1) to form a parallel four-helix bundle can be predicted from the SNAP-23N sequence using rules proposed by Harbury
et al. (27). An enrichment of -branched residues at the
d position but not the a position favors
coiled-coil tetramers, whereas the reverse pattern favors coiled-coil
dimers. Each intermolecular layer of our structure has the same four
hydrophobic side chains as a result of the 4-fold symmetry, whereas
other SNARE complex structures have different hydrophobic side chains
in each layer. The latter is also true for the syntaxin-1a
homotetrameric structure, because the chains in this complex are
antiparallel (26). Bulky hydrophobic side chains are accommodated by
complementary smaller hydrophobic ones in each layer of these other
SNARE complex structures (4). The SNAP-23N sequence lacks bulky
hydrophobic side chains that would clash with one another as a result
of parallel packing. Therefore, SNAP-23N may be unique among SNARE
coiled-coil domains in its ability to form a parallel homotetrameric complex.
The SNAP-23N structure has buried glutamines in the 0 layer like other SNARE complexes (7, 24, 26). Buried polar residues at the center of coiled-coil complexes are thought to be important for orienting the direction of coiled-coil complexes (28, 29). The molecular arrangement of the 0 layer varies among SNARE complex structures. The three buried glutamines in the neuronal heterotetrameric complex structure are stabilized by hydrogen bond contacts between their carbonyl groups and the guanidinium side chain of an arginine from VAMP-2 (7). A network of water molecules coordinates the four buried glutamines in the SNAP-25N/syntaxin-1a tetrameric complex (24). The buried glutamines in the syntaxin-1a homotetramer hydrogen bond directly to each other as in our structure (26). However, in contrast to the SNAP-23N structure, the four glutamines in the syntaxin-1a structure are arranged in pairs on opposite sides of the complex due to the antiparallel topology of the helices. Therefore, the hydrogen bonding arrangement of the four buried glutamines in the SNAP-23N homotetramer is a new variant to the SNARE complex 0 layer (Fig. 5B).
Finally, the surface of the SNAP-23N structure is mostly acidic like other SNARE complexes (Fig. 4C) (7, 24, 26). It was speculated that SNARE regulatory molecules, NSF and SNAPs, have diverse recognition properties for different SNARE complexes by surface complementarity rather than for a specific sequence (30).
The biological significance of the homotetramer we observe is unclear. To date, homooligomerization of SNAP-23 has not been demonstrated in vivo. However, SNARE complex assembly is difficult to study in cells, and therefore purified SNARE proteins are used as a surrogate for this process (23, 31). What is the conformation of a v-SNARE or t-SNARE prior to vesicle fusion? One possibility is that SNAREs are disordered on membranes before interacting to form a membrane fusion complex. On the other hand, this seems unlikely given the growing data base of assembly intermediate structures. Furthermore, the high local concentrations of SNARE proteins on membranes should favor oligomerization. We propose that the SNAP-23N homotetramer and other assembly intermediates may represent low stability "storage forms." This could prevent proteolytic degradation of SNAREs yet allow rapid reorganization into high affinity heterotetrameric "fusion-active" forms. Future studies will be necessary to demonstrate SNARE complex intermediates within cells.
In summary, we have shown that SNAP-23N forms a heterotetrameric
complex with other non-neuronal SNARE coiled-coil domains and,
independently, self-associates to form a parallel homotetramer. The
homotetrameric SNAP-23N structure has common and unique features compared with other reported SNARE complex structures and extends our
understanding of SNARE coiled-coil complex diversity and SNAP-23 function.
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ACKNOWLEDGEMENTS |
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We thank Amira Klip, Pietro De Camilli, and Richard Scheller for providing cDNA clones of human SNAP-23, rat VAMP-3, and rat syntaxin-4, respectively. We also thank Anand Saxena and Robert Sweet for help with x-ray data collection at beamline X-12C of the National Synchrotron Light Source of Brookhaven National Laboratories and Robert Flaumenhaft and Steven Blacklow for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NHL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Supported by a National Institutes of Health (NIH) K08 award from the NHLBI, NIH and an ASH Scholar Award from the American Society of Hematology. To whom correpondence should be addressed: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, 41 Avenue Louis Pasteur, Boston, MA 02115. Tel.: 617-667-0719; Fax: 617-667-2030; E-mail: sfreedm2@caregroup.harvard.edu.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M210483200
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ABBREVIATIONS |
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The abbreviations used are: SNARE, SNAP receptor; SNAP, soluble NSF attachment protein; NSF, N-ethylmaleimide-sensitive fusion protein; v-SNARE, vesicle SNARE; t-SNARE, target SNARE; SNAP-25, synaptosome-associated protein of 25 kDa; SNAP-23, SNAP-25-like protein of 23 kDa; VAMP, vesicle-associated membrane protein; MAD, multiwavelength anomalous diffraction; SeMet, selenomethionine; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.
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