From the Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, California 94305
Received for publication, November 21, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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The structure of a truncated SNARE complex has
been solved to 1.4-Å resolution revealing a stabilizing salt bridge,
sites of hydration, and conformational variability of the ionic
central layer that were not observed in a previously published
structure at 2.4-Å resolution (Sutton, R. B., Fasshauer, D.,
Jahn, R., and Brunger, A. T. (1998) Nature 395, 347-353). The truncated complex lacks residues involved in
phospholipid binding and denatures at a lower temperature than
longer complexes as assessed by SDS and circular dichroism
thermal melts. The truncated SNARE complex is monomeric, and it retains
binding to synaptotagmin I.
Members of the conserved family of
SNARE1 proteins play an
important role in protein-assisted vesicle membrane fusion (1-7). SNARE complex formation juxtaposes synaptic vesicle and plasma membranes and thus may set the stage for vesicle membrane fusion. In
the final stages of fusion, neurotransmitter release is probably regulated by the Ca2+-binding protein synaptotagmin (8).
Each SNARE protein contains at least one core domain that binds to
other SNARE proteins to form a four-helix bundle (8). This four-helix
bundle is composed of 16 layers transverse to the helical axes
including a buried ionic layer at the center of the four-helix bundle
(9).
The neuronal SNARE complex consists of three SNAREs: synaptobrevin,
syntaxin, and SNAP-25
(Synaptosome-associated
protein, 25 kDa) (Fig. 1). Synaptobrevin (also referred to
as vesicle-associated membrane protein) is a 12-kDa protein with
a SNARE binding domain and a single spanning transmembrane domain (10,
11). Syntaxin is a 35-kDa protein with a three-helix bundle regulatory
domain, a SNARE binding domain, and a single spanning transmembrane
domain (1, 3, 12, 13). SNAP-25 is a 25-kDa protein with two SNARE
binding domains and a linker domain of ~45 amino acids. SNAP-25 is
targeted to the plasma membrane by its association with syntaxin via
palmitoylation of three cysteine residues in the linker domain (14,
15).
The crystal structure of the neuronal SNARE complex revealed a
conserved buried ionic layer at the center of the four-helix bundle (9)
whose function is still uncertain (16). Most probably, it plays a role
during N-ethylmaleimide-sensitive factor (NSF) driven
disassembly of the SNARE complex, because mutations of this central
layer can disrupt this process (17).
Here we present the crystal structure of the neuronal SNARE complex at
a 1.4-Å resolution. To obtain this high resolution crystal structure,
the individual SNAREs were truncated in comparison with the
corresponding constructs used in the previously published crystal
structure solved at a 2.4-Å resolution (9). This high resolution structure reveals new sites of hydration and stabilizing intermolecular interactions. We further characterize the thermal stability of this SNARE complex by CD and SDS melts, its
oligomerization state, and its binding properties to synaptotagmin in
the presence of Ca2+ and EDTA.
Constructs
Constructs encoding sequences for the "minimal"
complex (Fig. 1), rat syntaxin 1a residues 180-262 (SXa),
synaptobrevin II residues 1-96 (SBa), SNAP-25 B residues 1-83 (SN1a),
and SNAP-25 B residues 120-206 (SN2a) were described elsewhere (18).
The cDNA-encoding sequences for the N-terminally truncated minimal complex and the microcomplex (Fig. 1), rat syntaxin 1a residues 188-262 (SXb) and residues 191-256 (SXc), synaptobrevin II residues 25-96 (SBb) and residues 28-89 (SBc), SNAP-25 B residues 7-83 (SN1b), and SNAP-25 B residues 132-204 (SN2b) and 141-204 (SN2c) were
subcloned from these constructs into the expression plasmid pET28a
(Novagen) or pGEX-2T (Amersham Biosciences) (SN2b only). The cDNA
encoding the sequence for rat synaptotagmin I-(139-421) were
subcloned from synaptotagmin I cDNA into the pGEX-2T expression vector. The G374 sequence variant of synaptotagmin (19) was generated
using the QuikChange mutagenesis kit (Stratagene) using the
oligomers 5'-TGTAACCAACGAAGACTTTGCCGATGGCGTCGTTCTTGCC-3' and 5'-GGCAAGAACGACGCCATCGGCAAAGTCTTCGTTGGTTACA-3'. The correct sequences of all of the constructs were verified by DNA sequencing (Biocore Inc.,
Palo Alto, CA, or Keck facility, Yale University, New Haven, CT).
The pET28a expression plasmids were transformed into E. coli
BL21(DE3) competent cells using standard protocols (20). Cells were
grown at 37 °C in a BIOFLO 3000 fermentor (New Brunswick, NJ) using
ECPYM1 medium (21) in the presence of 50 µg/ml kanamycin sulfate. The expression was induced with 1 mM
isopropyl-1-thio- Cells containing histidine-tagged SNARE proteins were resuspended in
1:10 denaturing lysis buffer (7 M guanidine, 50 mM Tris, pH 8.2, 10 mM imidazole) and passed
once through a Microfluidizer (Microfluidics) at 25,000 p.s.i.
Cell lysate was cleared by ultracentrifugation in a Beckman Optima
XL-100K Centrifuge using a type 45 rotor at 45,000 rpm for 45 min.
Cleared lysate was loaded in bulk for 10 h onto 25-ml
nickel-nitrilotriacetic acid resin (Qiagen). The column was washed with
10-column volumes of denaturing wash buffer (6 M guanidine,
50 mM Tris, pH 8.2, 20 mM imidazole) followed by 10-column volumes of native buffer (300 mM NaCl, 20 mM Tris, pH 7.7, 20 mM imidazole). The protein
was then eluted with 3-column volumes of native buffer containing
imidazole at 250 mM.
Cells containing glutathione S-transferase (GST)-tagged
synaptotagmin I C2AB and SN2b were resuspended in
300 mM NaCl, 50 mM
NaH2PO4, 50 mM Tris, 1 mM EDTA, 5 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 10 mM benzamidine, and 100 mg/ml DNase. Cells were lysed by
disruption using a Microfluidizer with two passes at 25,000 p.s.i. Cell
lysate was cleared by ultracentrifugation in a Beckman Optima XL-100K
Centrifuge using a type 45 rotor at 45,000 rpm for 45 min. Cleared
lysate was loaded in bulk for 4 h onto 25 ml of glutathione resin
(Amersham Biosciences) and washed with 10-column volumes of buffer.
Protein was eluted with 15 mM glutathione and dialyzed
overnight into 200 mM NaCl, 20 mM HEPES pH 7.8, 5 mM DTT, and 1 mM EDTA. Synaptotagmin was
further purified using fast protein liquid cation exchange
chromatography over a Mono S 10/10 column (Amersham Biosciences).
The concentration of the proteins was calculated by using a Bradford
assay or UV absorption at 280 nm. UV spectra demonstrated that
synaptotagmin was free of the DNA contamination mentioned elsewhere
(19).
The SNARE complex was formed by mixing SNAP-25, synaptobrevin, and
syntaxin fragments at a 1:1:1:1 ratio, mixed with 4 M urea to prevent precipitation, and dialyzed into 100 mM NaCl, 20 mM Tris, pH 8.2, and 1 mM CaCl2 at
4.0 °C. Thrombin was added after 4 h to remove the histidine
tags from the proteins, and dialysis was continued for 10 h. SNARE
complex was purified on a Mono Q 10/10 anion exchange column
where it eluted between 230 and 330 mM NaCl. Final size
exclusion purification was performed using a Superdex 200 16/60 column
(Amersham Biosciences).
The purity of the SNARE complex was assayed by SDS-PAGE using
Phast gels (Amersham Biosciences) and Coomassie Blue staining. Final
fractions containing completely formed SNARE complex were pooled, and
the protein concentration was determined by UV absorption at 280 nm.
The complex was flash-frozen in liquid nitrogen and stored at
Biochemistry
CD--
CD experiments were performed on a Aviv 62DS
spectrometer at 150 mM NaCl, 20 mM
Na2HPO4, pH 7.8, and 10 µM SNARE
complex. Temperature scans were performed between 37 and 97 °C at
two-degree intervals with 1-min equilibration between temperature
changes and 1-min acquisitions with data averaging at each temperature point.
GST Pull-down Assays--
GST pull-down experiments were
performed at room temperature in 150 mM NaCl with 20 mM HEPES, pH 7.8, and 1 mM DTT in the presence
of either 0.5 mM CaCl2 or 1 mM
EDTA. Samples were mixed at room temperature, incubated for 1 h
with GST beads, and washed three times with buffer and mixed with SDS
sample loading buffer. The samples were analyzed by SDS-PAGE using
10-25% SDS Phast gels.
Multi-angle Laser Light Scattering (MALLS)--
Size exclusion
chromatography was performed using a Superdex 200 10/30 column at a
flow rate of 0.5 ml/min. Measurements were performed in 150 mM NaCl, 10 mM HEPES, pH 7.8, and 5 mM DTT. The elution profile was monitored by UV absorption
at 280 nm, light scattering at 690 nm, and differential refractometry.
Light scattering and differential refractometry were carried out using
the Dawn and OptiLab instruments (Wyatt Technology). Analysis was
carried out using the Astra software (22). For each sample, 100 µl of protein at 1 mg/ml protein was loaded. The differential
refractive index increment (dn/dc) is fairly constant for proteins and
was set to 0.185.
Crystallography
Crystallization--
Crystallization trials were conducted using
the hanging drop vapor diffusion method. The initial SNARE protein
concentration was 9 mg/ml in a solution of 200 mM NaCl,
10 mM HEPES (Fluka), pH 7.8, and 5 mM
DTT (American BioAnalytical). Crystals appeared at 4 °C in 1-3 days
and grew to full size in 3-5 days. The well solution contained
15-20% (±)-2-methyl-2,4-pentanediole (MPD) (Fluka), 75-125
mM CaCl2 (Fluka), and 50 mM MES
(Fluka) at pH 5.0-6.0. Initial drops consisted of a one-to-one mixture
of protein sample and well solution resulting in a total volume of 4 µl. The crystals grew in clusters as thick needles. To obtain single crystals, these clusters of crystals were used for streak seeding into
preequilibrated hanging drops. Single crystals were prepared for
freezing by serial transfer using nylon loops into mother liquor with
increasing amounts of MPD as a cryoprotectant up to 55%. Crystals were
then frozen by rapid transfer directly into liquid nitrogen.
Diffraction Data--
Diffraction data were collected at the
Lawrence Berkeley National Laboratory Advanced Light Source beamline BL
8.2.1 from a single crystal in one pass at 100 K using an Area Detector
System Quantum 210 2 × 2 CCD detector. The diffraction data were
collected to a 1.4-Å resolution. All of the data processing was
carried out using the programs Denzo and Scalepack (23). Statistics of
the diffraction data are shown in Table I. The crystals formed with one
copy of the SNARE complex per asymmetric unit in space group
P212121.
Phases--
The phases for the diffraction data were
obtained by molecular replacement using the direct rotation search (24)
as implemented in the program CNS (version 1.1) (25) using diffraction
data from 20 to 3.5-Å resolution. The subsequent translation function used diffraction data from 15 to 4.0-Å resolution and resulted in an
unambiguous solution. The search model consisted of one of the three
non-crystallographically related copies of the neuronal SNARE solved at
2.4 Å (9). It was truncated to contain only those residues present in
the microcomplex.
Model Building--
Model building was performed using the
program O (26). The initial model was optimized by rigid body
refinement followed by simulated annealing with torsion angle dynamics
(27), restrained B-value refinement (28), and conjugate
gradient minimization using the MLF target function (29).
Overall anisotropic scale factors and bulk solvent correction were
applied to the diffraction data. The progress of model rebuilding and
refinement was monitored by cross-validation using
Rfree (30), which was computed from a randomly
chosen test set comprising 10% of the data. The sites of hydration
were placed by inspection of peaks larger than three standard
deviations above the mean in Fo Oligomeric State of the SNARE Complex--
The neuronal SNARE
complex has a tendency to oligomerize as shown by analytical
ultracentrifugation and MALLS (18). The minimal SNARE complex
obtained by limited proteolysis that was used in the 2.4-Å crystal
structure had an apparent molecular mass of 60-90 kDa compared with a
calculated molecular mass of 41 kDa (18). The C-terminal
truncations of synaptobrevin by botulinum toxin B or tetanus toxin
produced a monomeric SNARE complex (34). Furthermore, the C-terminal
truncation of endobrevin (vesicle-associated membrane protein 8) in the
endosomal SNARE complex produced a monodisperse sample (35). Therefore,
we truncated the neuronal synaptobrevin at Trp-89 along with the
appropriate truncations of syntaxin and SNAP-25 (Fig.
1). These truncations resulted in the
removal of approximately one SNARE Complex Stability--
We performed
temperature-dependent SDS and CD melts of the micro-SNARE
complex and compared the results to both the minimal SNARE complex (36)
and a SNARE complex that was obtained from the minimal complex by
truncation at the N terminus (Fig. 2). Our experiments revealed a roughly 20 °C reduction in the stability of the micro-SNARE complex in SDS relative to both the minimal and
N-terminally truncated SNARE complexes (Fig. 2a). In light of this reduction in SDS stability, we further investigated the thermal
stability of the various SNARE complexes under native conditions by CD.
Both the minimal and the N-terminally truncated SNARE complexes have a
Tm of 94 °C, whereas the micro-SNARE complex has a reduced Tm of 89 °C (Fig.
2b).
Synaptotagmin Binding--
Having demonstrated that the
micro-SNARE complex forms a quantitative and stable complex, albeit
with somewhat reduced Tm, we investigated whether it
would retain its ability to interact with the C2 domains of
synaptotagmin I. GST pull-down experiments were conducted in the
presence of both 1 mM EDTA and 0.5 mM
CaCl2. As shown in Fig. 3,
synaptotagmin I is capable of binding the micro-SNARE complex in both
the presence and absence of Ca2+. These findings are
consistent with prior reports using the C2AB domain of
synaptotagmin III and the minimal SNARE complex (37).
Microcomplex Structure--
We next determined the crystal
structure of the microcomplex. Crystals were obtained in space group
P212121 in the presence of MPD and
CaCl2 at 4 °C. These conditions are similar to the previous crystallization conditions used for the minimal SNARE complex
(9). The crystal structure contained only one copy of the complex per
asymmetric unit in contrast to the minimal SNARE complex that
crystallized in a different space group (I222) with three complexes per
asymmetric unit. Most importantly, the crystals of the microcomplex
diffracted to 1.4 Å, making this the highest resolution crystal
structure of a SNARE complex available to date. All of the residues of
the microcomplex were visible in the final model, which refined to a
Rcryst value of 19.8% and a
Rfree value of 22.4%. The statistics
of the diffraction data and the final
refined model are shown in Tables I and
II. The electron density maps are of
excellent quality (Fig. 5b) and allowed assignments
of nearly all of the side-chain rotamers.
Three Ca2+ sites were found that are coordinated by
symmetry-related molecules. These sites were visible as 8
As expected, the micro-SNARE complex forms a four-helix bundle. The
C
To further compare the various SNARE complex crystal structures, we
superimposed the residues around the ionic central layer (Fig. 1) with
the corresponding residues of the previously solved structures. For the
neuronal SNAREs, the layer consists of synaptobrevin Arg-56, syntaxin
Gln-226, SNAP-25 Gln-53, and SNAP-25 Gln-174. For the endosomal
complex, the corresponding residues are endobrevin Arg-76, syntaxin-7
Gln-199, vti1b Gln-170, and syntaxin-8 Gln-179. The root mean square
differences for residues at this layer between the microcomplex and the
endosomal complex are 0.315 and 0.509 Å for C
The quality of our diffraction data allowed us to assign numerous sites
of hydration that were previously unobservable (Fig. 5b). Of
particular interest is a buried water molecule (Fig. 5b, H2O 89) at the ionic central layer. This
water molecule is located 3.10 Å from the
The formation of salt bridges on the surface of proteins is known to
stabilize exposed structural elements. The presence of surface salt
bridges positioned to stabilize buried structural elements is less
common. However, a carboxylic acid The neuronal SNARE complex can form oligomers under various
conditions (18, 44). We have shown that C-terminal truncation of
synaptobrevin, C-terminal to residue 89 (see Fig. 1), along with
C-terminal truncation of syntaxin produces a SNARE complex that is both
monomeric and monodisperse. Interestingly, the fragments of
synaptobrevin containing residues 77-90 bind to phospholipids (40,
41). Thus, it is possible that some of the residues that are involved
in phospholipids binding are also involved in oligomerization.
We have shown that C-terminal truncation but not N-terminal truncation
of the minimal SNARE complex results in a reduction in stability as
measured by CD and SDS thermal melts. Other investigators have used SDS
stability to characterize various combinations of SNARE proteins (42).
Thus, considering the influence on stability by the C-terminal end of
the SNARE complex, a comparison of SDS stability should be viewed with
caution when comparing the stabilities of various SNARE combinations.
Our finding that the microcomplex retains the ability to bind
synaptotagmin is particularly noteworthy in view of the recent discovery of DNA contaminants and sequence mutations in earlier binding
studies with synaptotagmin I C2AB (19). Thus, SNARE binding
by synaptotagmin is independent of oligomerization of the SNARE complex.
Through C-terminal truncation, we obtained a microcomplex that allowed
us to solve the structure of the SNARE complex at near atomic
resolution. The structure confirmed many of the observations that we
reported in our previous 2.4-Å structure (9). The structure also
revealed several new details of the SNARE complex. The existence of
alternate conformations of the buried arginine at the ionic central
layer is consistent with a function for this layer in NSF-mediated
disassembly of the SNARE complex (17). Previously, unobservable
interactions included a buried water molecule and a salt bridge that
were likely to play an important role in stabilizing the ionic central
layer (Fig. 5b). Such structural details offer important new
information for the design of future experiments to study SNARE complex function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside at an
A600 of 20. The pGEX-2T expression
plasmids were transformed into E. coli BL21-competent cells
using standard protocols. Cells were grown at 37 °C in terrific
broth supplemented with 100 µg/ml ampicillin media in 4-liter flasks.
At an A600 of ~1, the temperature was reduced
to 25 °C and expression was induced for 3 h using 1 mM isopropyl-1-thio-
-D-galactopyranoside.
Approximately 3 h after induction, cells were harvested by
centrifugation for 20 min at 4200 rpm in a Beckman J6-HC
Centrifuge using a JS-4.2 rotor. Cells were immediately frozen
in liquid nitrogen and stored at
80 °C.
80 °C.
Fc
A-weighted electron density maps.
Only those sites were kept that exhibited reasonable protein solvent
hydrogen-bonding distances without steric conflict and whose
B-value refined to <55 Å2. MPD and Ca2
+ were identified by inspection of Fo
Fc and 2Fo
Fc
A-weighted electron density maps. At various points during refinement,
A-weighted,
annealed 2Fo
Fc composite omit
maps were used to minimize the effects of model bias. All of the
refinements were carried out using the program CNS (25). Statistical
linear least-squares superposition of the structures was performed
using the LSQMAN (31) from the Uppsala software factory suite.
Graphical images were prepared using PyMOL (Fig. 5) (32) or GRASP (Fig.
4, a and b) (33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical turn at the C-terminal ends of
syntaxin and synaptobrevin (Fig. 1). The truncated neuronal SNARE
proteins were then expressed, purified, and assembled. This
"micro"-SNARE complex has an apparent molecular mass of 32.5 kDa ± 2% as determined by MALLS (data not shown) compared with a
calculated molecular mass of 32.5 kDa. Thus, the microcomplex is both
monomeric and monodisperse.
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Fig. 1.
Sequence alignment of neuronal
SNAREs. The sequence for the constructs used for the
2.4-Å crystal structure of the minimized complex (9) is shown in
italics. In text, they are referred to as constructs
SBa, SXa, SN1a, and SN2a. The N-terminal truncations of these
constructs are underlined. In text, they are referred to as
constructs SBb, SXb, SN1b, and SN2b. The constructs of the microcomplex
are indicated by boxes. In the text, these constructs are
referred to as SBc, SXc, SN1b, and SN2c. Hydrophobic layers of the
SNARE complex are shown in blue, and the ionic central
(zero) layer is shown in red. Sequences displayed
are rat synaptobrevin II (gi:6981613), rat syntaxin-1A (gi:207126), rat
SNAP-25 B (gi:2116627).
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Fig. 2.
Stability of SNARE complexes.
A, SDS stability of the minimal complex, the N-terminally
truncated minimal complex, and the microcomplex. Experiments
were performed as described previously (43). SNARE complex was mixed
with SDS to a final concentration of 0.67%, heated at the indicated
temperature for 5 min, and immediately run on a 10-15% SDS-PAGE gel.
B, CD thermal melts of SNARE complexes as monitored at 220 nm. The minimal SNARE complex is shown in blue, the
N-terminal truncation of the minimal SNARE complex is shown in
red, and the microcomplex is shown in
green.
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Fig. 3.
Synaptotagmin I binding of microcomplex.
Shown is a GST pull-down assay of the micro-SNARE complex using the
GST-tagged C2AB fragment of synaptotagmin I (residues
139-421). In each lane, 2 µM microcomplex was incubated
with 2.0 µM GST-synaptotagmin I C2AB
(lanes 1 and 2) or 2 µM GST alone
(lanes 3 and 4) and glutathione resin.
Experiments were performed in the presence of 0.5 mM
Ca2+ (lanes 1 and 3) or 1 mM EDTA (lanes 2 and 4). The
microcomplex is indicated by an asterisk, and
GST-synaptotagmin is indicated by an arrow.
Data statistics
Refinement statistics
peaks in
2Fo
Fc maps. The coordinating
oxygen atoms are located on SNAP-25 Gln-20 and Glu-27 of a SNARE
complex and synaptobrevin Asp-80, Lys-83 of SNAP-25, Tyr-88, Trp-89,
and syntaxin Lys-256 of a symmetry-related complex. Several water
molecules complete the coordination spheres around the
Ca2+. Because these Ca2+ sites are located at
the artificially truncated C terminus of the microcomplex, it is
probable that these binding sites are the result of crystallization conditions.
atoms of the microcomplex were superimposed on that of
the minimal complex structure and on all of the homologous residues of
the endosomal complex structure. The results of these superpositions
are shown in Fig. 4, a and
b, and Table III. It is
interesting to note that the root mean square (r.m.s) difference between the microcomplex structure and the endosomal structure is
larger than the root mean square difference observed when comparing either structure to the minimal complex structure. In contrast to the
endosomal structure, the microcomplex displays little variation in
B-values over most of the four-helix bundle (see Fig.
4c). Only the second
-helix of SNAP-25 between layers 2 and 8 and synaptobrevin between layers
7 and 0 display any systematic
increase in C
B-values.
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Fig. 4.
Superposition of neuronal and endosomal SNARE
complex. A, C superposition of
the micro-SNARE complex and the endosomal SNARE complex (Protein Data
Bank code 1GL2). The color code is as follows: synaptobrevin is
shown in blue; syntaxin is shown in red; SNAP-25
is shown in green; endobrevin is shown in light
blue; vti1b is shown in magenta; and syntaxin-8 and
syntaxin-7 are shown in yellow. B,
C
superposition of the micro-SNARE complex with the
three complexes in the 2.4-Å crystal structure of the minimal SNARE
structure (Protein Data Bank code 1SFC). The color code is as follows:
the microcomplex is shown in red; the first molecule of the
minimal complex crystal structure (chains a-d) (see Ref. 9) is shown
in dark blue; the second molecule (chains e-h) is shown in
medium blue; and the third molecule (chains i-l) is shown
in light blue. C, B-value plot for the
C
residues of the micro-SNARE complex. The color code is
as follows: synaptobrevin is shown in blue; syntaxin is
shown in red; SNAP-25 SN1c is shown in light
green; and SNAP-25 SN2c is shown in dark green.
C superpositions
and all
atoms, respectively. Several of the SNARE crystal structures show the
presence of a bifurcated hydrogen bond between synaptobrevin Arg-56 and
SNAP-25 Gln-53 and Gln-174 (Fig.
5a). However, in one of the
molecules of the minimal complex crystal structure (Fig. 5a,
cyan), Arg-56 exhibits a rotamer that allows direct hydrogen
bonding from each of the side-chain nitrogen atoms of Arg-56 to each of
the buried glutamines. This Arg-56 rotamer is also visible in the
structure of the squid neuronal SNARE complex with complexin (38).
Thus, Arg-56 exhibits significant conformational variability among the
different structures, whereas the three glutamines exhibit very similar
conformations (Fig. 5a). The observed conformational
variability of the central layer may suggest a possible functional role
in the disassembly process (39).
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Fig. 5.
Ionic central layer. A, alternate
conformations of synaptobrevin Arg-56 at the ionic central layer.
Residues for the microcomplex structure are shown in yellow,
residues for the minimal complex (chains a-d) are shown in
cyan, and residues for the endosomal structure are shown in
gray. Residues are numbered according to the sequences of
the neuronal SNARE complex. B, electron density at the ionic
central layer of the micro-SNARE complex. Shown is a
2Fo Fc
A-weighted
omit map. The electron density is contoured at 1.5
. Synaptobrevin
is shown in blue, syntaxin is shown in red, and
SNAP-25 is shown in green.
-nitrogen of Arg-56,
satisfying the hydrogen bond requirements of this nitrogen. It is
possible that this water molecule is not present when Arg-56 adopts
alternate conformations observed in some of the other crystal structures.
-oxygen of SNAP-25 Glu-170 shows
just such an interaction buttressing SNAP-25 Gln-174 at 2.79 Å through
the Gln-174
-nitrogen (Fig. 5b). This interaction
stabilizes the SNAP-25 Gln-174
-oxygen, which in turn interacts with
synaptobrevin Arg-56 N
2 at a distance of 2.65 Å (Fig.
5b). The close interaction of synaptobrevin Arg-56 N
2
with the SNAP-25 Gln-174
-oxygen as compared with the Arg-56 N
1
and syntaxin Gln-226 interaction probably reflects the proximity of the
negatively charged carboxylic group from SNAP-25 Glu-170. We note
further that SNAP-25 Glu-170 is a highly conserved residue in the
SNAP-25 family, including the endosomal SNARE syntaxin-8.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank B. S. DeLeBarre for assistance with MALLS, B. S. DeLeBarre, M. E. Bowen, S. Fukai, J. Hyman, E. H. Panepucci, and R. B. Sutton for helpful discussions and critical reading of the paper. Diffraction data were collected at beamline BL 8.2.1 at the Lawrence Berkeley National Laboratory Advanced Light Source. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, and Materials Sciences Division of the U. S. Department of Energy under Contract number DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.
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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 1N7S) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.: 650-736-1031;
Fax: 650-745-1463; E-mail: brunger@stanford.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211889200
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ABBREVIATIONS |
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The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SNAP-25, Synaptosome-associated protein, 25 kDa; GST, glutathione S-transferase; DTT, dithiothreitol; MALLS, multi-angle laser light scattering; MPD, (±)-2-methyl-2,4-pentanediole; MES, 4-morpholineethanesulfonic acid.
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1. | Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418[Medline] [Order article via Infotrieve] |
2. | Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Söllner, T. H., and Rothman, J. E. (1998) Cell 92, 759-772[Medline] [Order article via Infotrieve] |
3. | Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259[Medline] [Order article via Infotrieve] |
4. | Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263, 1146-1149[Medline] [Order article via Infotrieve] |
5. | Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., De Camilli, P., Südhof, T. C., Niemann, H., and Jahn, R. (1993) Nature 365, 160-163[CrossRef][Medline] [Order article via Infotrieve] |
6. | Ferro-Novick, S., and Jahn, R. (1994) Nature 370, 191-193[CrossRef][Medline] [Order article via Infotrieve] |
7. | Südhof, T. C. (1995) Nature 375, 645-653[CrossRef][Medline] [Order article via Infotrieve] |
8. | Fernández-Chacón, R., Königstorfer, A., Gerber, S. H., Garcia, J., Matos, M. F., Stevens, C. F., Brose, N., Rizo, J., Rosenmund, C., and Südhof, T. C. (2001) Nature 410, 41-49[CrossRef][Medline] [Order article via Infotrieve] |
9. | Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature 395, 347-353[CrossRef][Medline] [Order article via Infotrieve] |
10. | Trimble, W. S., and Scheller, R. H. (1988) Trends Neurosci. 11, 241-242[Medline] [Order article via Infotrieve] |
11. | Baumert, M., Maycox, P. R., Navone, F., De Camilli, P., and Jahn, R. (1989) EMBO J. 8, 379-384[Abstract] |
12. | Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell 74, 863-873[Medline] [Order article via Infotrieve] |
13. | Fernandez, I., Ubach, J., Dulubova, I., Zhang, X., Südhof, T. C., and Rizo, J. (1998) Cell 94, 841-849[Medline] [Order article via Infotrieve] |
14. | Oyler, G. A., Higgins, G. A., Hart, R. A., Battenberg, E., Billingsley, M., Bloom, F. E., and Wilson, M. C. (1989) J. Cell Biol. 109, 3039-3052[Abstract] |
15. |
Vogel, K.,
Cabaniols, J. P.,
and Roche, P. A.
(2000)
J. Biol. Chem.
275,
2959-2965 |
16. |
Wei, S.,
Xu, T.,
Ashery, U.,
Kollewe, A.,
Matti, U.,
Antonin, W.,
Rettig, J.,
and Neher, E.
(2000)
EMBO J.
19,
1279-1289 |
17. |
Scales, S. J.,
Yoo, B. Y.,
and Scheller, R. H.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
14262-14267 |
18. | Fasshauer, D., Eliason, W. K., Brunger, A. T., and Jahn, R. (1998) Biochemistry 37, 10354-10362[CrossRef][Medline] [Order article via Infotrieve] |
19. | Ubach, J., Lao, Y., Fernandez, I., Arac, D., Südhof, T. C., and Rizo, J. (2001) Biochemistry 40, 5854-5860[CrossRef][Medline] [Order article via Infotrieve] |
20. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1998) in Current Protocols in Molecular Biology (Chanda, V. B., ed), Vol. 1 , pp. 1.1-1.8, John Wiley & Sons, Inc., New York |
21. | Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T. (2000) Current Protocols in Protein Science , Vol. 1 , pp. 5.1-5.3, John Wiley & Sons, Inc., New York |
22. | Wyatt, P. (1993) Anal. Chim. Acta 272, 1-40[CrossRef] |
23. | Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-325 |
24. | DeLano, W. L., and Brunger, A. T. (1995) Acta Crystallogr. Sec. D 51, 740-748[CrossRef][Medline] [Order article via Infotrieve] |
25. | Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sec. D 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
26. | Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sec. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
27. | Rice, L. M., and Brunger, A. T. (1994) Proteins 19, 277-290[Medline] [Order article via Infotrieve] |
28. | Hendrickson, W. A. (1985) Methods Enzymol. 115, 252-270[Medline] [Order article via Infotrieve] |
29. |
Adams, P. D.,
Pannu, N. S.,
Read, R. J.,
and Brunger, A. T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5018-5023 |
30. | Brunger, A. T. (1992) Nature 355, 472-475[CrossRef] |
31. | Kleywegt, G. J. (1996) Acta Crystallogr. Sec. D 52, 842-857[CrossRef] |
32. | DeLano, W. L. (2002) The PyMOL Molecular Graphics System, version 0.8 , DeLano Scientific, San Carlos, CA, (www.pymol.org) |
33. | Nicholls, A., Sharp, A., and Honig, B. (1991) Proteins Struct. Funct. Genet. 11, 281-296[Medline] [Order article via Infotrieve] |
34. |
Margittai, M.,
Fasshauer, D.,
Pabst, S.,
Jahn, R.,
and Langen, R.
(2001)
J. Biol. Chem.
276,
13169-13177 |
35. |
Antonin, W.,
Holroyd, C.,
Tikkanen, R.,
Honing, S.,
and Jahn, R.
(2000)
Mol. Biol. Cell
11,
3289-3298 |
36. |
Fasshauer, D.,
Sutton, R. B.,
Brunger, A. T.,
and Jahn, R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15781-15786 |
37. |
Sutton, R. B.,
Ernst, J. A.,
and Brunger, A. T.
(1999)
J. Cell Biol.
147,
589-598 |
38. |
Bracher, A.,
Kadlec, J.,
Betz, H.,
and Weissenhorn, W.
(2002)
J. Biol. Chem.
277,
26517-26523 |
39. |
Scales, S. J.,
Finley, M. F.,
and Scheller, R. H.
(2001)
Science
294,
1015-1016 |
40. |
Quetglas, S.,
Iborra, C.,
Sasakawa, N.,
De Haro, L.,
Kumakura, K.,
Sato, K.,
Leveque, C.,
and Seagar, M.
(2002)
EMBO J.
21,
3970-3979 |
41. |
Quetglas, S.,
Leveque, C.,
Miquelis, R.,
Sato, K.,
and Seagar, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9695-9700 |
42. | Foster, L. J., Yeung, B., Mohtashami, M., Ross, K., Trimble, W. S., and Klip, A. (1998) Biochemistry 37, 11089-11096[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Chen, Y. A.,
Scales, S. J.,
Duvvuri, V.,
Murthy, M.,
Patel, S. M.,
Schulman, H.,
and Scheller, R. H.
(2001)
J. Biol. Chem.
276,
26680-26687 |
44. |
Hua, Y.,
and Scheller, R. H.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
8065-8070 |