From the Departments of Structural Biology and of
§ Molecular and Cellular Physiology and
The Howard
Hughes Medical Institute, Stanford University School of Medicine,
Stanford, California 94305
Received for publication, October 23, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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Intracellular membrane fusion requires SNARE
proteins found on the vesicle and target membranes. SNAREs associate by
formation of a parallel four-helix bundle, and it has been suggested
that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A,
designated H3, contributes one of the four helices to the SNARE
complex. In the crystal structure of syntaxin 1A H3, four molecules
associate as a homotetramer composed of two pairs of parallel helices
that are anti-parallel to each other. The H3 oligomer observed
in the crystals is also found in solution, as assessed by gel
filtration and chemical cross-linking studies. The crystal structure
reveals that the highly conserved Phe-216 packs against
conserved Gln-226 residues present on the anti-parallel pair of
helices. Modeling indicates that Phe-216 prevents parallel tetramer
formation. Mutation of Phe-216 to Leu appears to allow formation of
parallel tetramers, whereas mutation to Ala destabilizes the protein.
These results indicate that Phe-216 has a role in preventing formation
of stable parallel helical bundles, thus favoring the interaction of
the H3 region of syntaxin 1a with other proteins involved in membrane fusion.
Eukaryotic cells transport cargo between different intracellular
compartments, and release selective cargo into the extracellular space.
This task requires that transport vesicles bud from the membranes of
organelles and fuse specifically with target membranes. An extensively
studied example of this process is synaptic vesicle exocytosis, in
which neurotransmitter-filled vesicles fuse with the plasma membrane of
a presynaptic neuron to release neurotransmitter into the synaptic
cleft (1). The process of directed docking and fusion of vesicles is
mediated by several proteins; these include the soluble
NSF1 attachment proteins
(SNAPs), the SNAP receptor proteins (SNAREs) NSF, Sec1 proteins,
and the Rab small GTPases. In neurons, the SNARE proteins involved in
this process are VAMP2, syntaxin 1a, and SNAP-25.
The SNARE proteins are believed to be directly involved in the
intracellular membrane fusion event. SNAREs are associated with vesicle
(v-SNARE) and target (t-SNARE) membranes and can interact with each
other by forming stable four-helix bundles (SNARE complexes). The
synaptic vesicle v-SNARE VAMP2 contains a small cytoplasmic domain
followed by a C-terminal transmembrane anchor, and the cytoplasmic
domain is largely unstructured in isolation (2, 3). The t-SNARE SNAP-25
is attached to the plasma membrane via palmitoylation of four cysteine
residues and displays a significant amount of disorder in isolation in
solution (2, 4). However, both VAMP2 and SNAP-25 adopt helical
conformations when part of the SNARE complex. The t-SNARE syntaxin 1a
consists of an N-terminal domain termed Habc, a region of ~9 kDa
termed H3, and a C-terminal transmembrane anchor. The Habc domain is a
three-helix bundle (5, 6), and the H3 region forms an amphipathic helix
that associates with either partner SNAREs or the Habc domain (7, 8).
The three SNARE proteins have a modest affinity for each other in
binary complexes (~1 µM), but the ternary
SNAP-25·VAMP2·syntaxin 1a SNARE complex is extremely stable
(9).
The x-ray crystal structure of the "core" SNARE complex, which
consists of the cytoplasmic portion of VAMP2, two fragments of SNAP-25,
and the H3 region of syntaxin 1a, reveals that these three proteins
form a 110-Å-long parallel four-helix bundle (7). Syntaxin 1a H3 and
VAMP2 each contribute one helix to the complex, and SNAP-25 contributes
two helices. When bound to the regulatory protein nSec1, syntaxin 1a
exists as a four-helix bundle (8). The N-terminal half of H3
contributes one helix to the bundle, and the C-terminal half consists
of a mixture of short helices and random coils. Because only the
H3 region participates in the ternary 7 S complex, it has been proposed
that syntaxin 1a must undergo large conformational changes to allow
Habc to dissociate from H3, and allow syntaxin 1a to bind VAMP2 and
SNAP-25 (10, 11). It has also been observed that syntaxin 1a can form
stable binary complexes with SNAP-25 in vitro and that these
complexes may be a precursor to SNARE complex assembly (4, 12).
Syntaxin 1a associates with many other proteins in the presynaptic cell (13-15), and it is possible that other conformations of the protein exist but have not yet been observed.
To better understand the role of syntaxin 1a in membrane fusion, we
have determined the x-ray crystal structure of the H3 region of
syntaxin 1a. The structure reveals that H3 forms a homotetramer, and
suggests that a conserved phenylalanine residue may determine the
orientation of the four helices with respect to each other. Mutation of
this phenylalanine to alanine or leucine confer different properties on
the H3 oligomeric assembly, as assessed through gel filtration
chromatography, chemical cross-linking, and circular dichroism
spectroscopy. The structure also suggests mechanisms for SNARE complex
assembly and may provide a model for SNARE precursor complexes.
Protein Expression and Purification--
The H3 region of
syntaxin 1a (residues 191-267) was cloned into the vector pGEX-KG
(Amersham Pharmacia Biotech) as described previously (16), and the
vector was transformed into the AB1899 strain of Escherichia
coli. The cells were grown in LB broth at 37 °C for ~1 h in
the presence of 1 mM ampicillin and then induced with 1 mM
isopropyl-1-thio- Crystallization--
Crystals of the wild type and Y257C
mercury-derivatized (see below) syntaxin 1a H3 region were obtained by
hanging drop vapor diffusion at 25 °C by mixing equal volumes of
purified protein in a buffer containing 300 mM NaCl and 15 mM Tris, pH 8.4, and reservoir solution containing 750 mM sodium/potassium tartrate, 100 mM sodium
acetate, pH 5.0-5.3. Crystals appeared within 1 week and grew to an
average size of 0.25 × 0.25 × 0.10 mm3 within 2 weeks. The crystals belong to space group P3221 and have
approximate unit cell dimensions a = b = 88.5 Å, c = 58.4 Å. The asymmetric unit contains
two copies of the H3 molecule with a 65% solvent content, which
represents half of the four-helix bundle. Typically, the crystals were
nonmosaic (~0.5°) and diffracted to an average resolution of 3.2 Å, however, the Y257C mutant protein crystals generally diffracted to
higher resolution (3.0-Å Bragg spacings). The diffraction patterns for
both the wild type and mutant proteins contained systematically weak
reflections, indicating the possible presence of multiple lattices or
pseudo-symmetry. A second crystal form, similarly packed but with a
doubled c axis (a = b = 88.5 Å, c = 115.9 Å) to give the enantiomorphic space group P3121, was also obtained. This crystal form was not
reproducible, but did provide the high resolution native data (2.4-Å
Bragg spacings) used for refinement. The asymmetric unit of this
crystal contains four copies of H3, corresponding to the entire
four-helix bundle.
Data Collection--
Diffraction data were measured from
crystals rapidly transferred from mother liquor to 30% ethylene glycol
and then flash-frozen at 100 K. Native and mercury derivative
diffraction data were measured on an R-AXIS IIC imaging plate detector
(Rigaku) using CuK Heavy Atom Derivative Production--
To obtain phases for
structure determination, selenomethionine-substituted protein was
prepared, but this protein failed to crystallize. Over 150 experiments
to prepare derivatives by soaking native crystals in many different
heavy atom compounds failed. It is likely that the low pH and high salt
conditions of the crystals prevented heavy atom binding, but the
crystals could not be adapted into significantly different conditions.
To combat this problem, single cysteine substitutions were introduced
into the H3 protein using the QuikChange kit (Stratagene) to allow for
covalent attachment of mercury atoms. Of 35 constructs containing
single-cysteine substitutions, 15 failed to yield significant amounts
of protein when expressed in BL21, JM109, or M15 E. coli
host strains. Purification and expression of the remaining 20 mutant
proteins proceeded as for the native protein. Only three of these
mutant proteins (K252C, V255C, and Y257C) crystallized, so these three
proteins were used for derivatization experiments. Crystals of the
mutant proteins were soaked in several different mercurial compounds,
but again binding was not observed. A successful strategy was to react
the purified mutant proteins in solution (12 h, 4 °C in a buffer of 300 mM NaCl, 15 mM Tris, pH 8.2) with mercury
compounds, and then apply the proteins to a gel filtration column
(Sephadex G15 resin) to remove unbound mercury compounds. Covalent
attachment of the mercury atom was assayed by mass spectrometry (data
not shown). The only successful heavy atom derivative was obtained by
reacting 2,6-bischloromercuri-4-nitrophenol with the Y257C mutant protein.
Phasing--
A difference Patterson synthesis calculated between
the 2,6-bischloromercuri-4-nitrophenol derivative of theY257C mutant
protein and native H3 data sets revealed a single mercury site.
Refinement of this site using the program
SOLVE2 gave SIRAS
phases to 3.3-Å Bragg spacings. The resulting electron density maps
revealed the helical backbone but were not of sufficient quality to
assign the sequence. A multiwavelength anomalous dispersion (MAD)
experiment was then carried out on the same derivatized crystal, and
phases to 2.7 Å were calculated using SOLVE with the edge
( Model Refinement--
Model building was done with the program O
(20). All refinement was carried out using the program CNS (21).
Interpretation of the electron density maps was complicated by the
location of the mercury atom that was used for phasing. The derivatized
cysteine is present in a disordered portion of the polypeptide chain,
but the mercury was bound in a surface pocket of a crystal lattice neighbor. Before refinement, a random subset (~8%) of the data was
removed and used as a test set for cross-validation. The model was
first subjected to simulated annealing with a maximum likelihood target (22) using amplitudes against the 2.7-Å mercury edge data set. After 5 cycles of refinement and manual rebuilding, the model
was refined against the 2.4-Å native data set with a maximum
likelihood target using amplitudes. In this form, the c axis
is doubled, so that the 2-fold axis that generates the tetramer is
noncrystallographic. Consideration of the relationship of the two
crystal forms allowed the partially refined model to be placed into the
larger unit cell by rigid-body refinement. Subsequent rounds of model
building using Chemical Cross-linking--
H3 protein was purified on a
Superdex 200 column HR 26/60 (Amersham Pharmacia Biotech) in 150 mM NaCl and 20 mM Tris, pH 8.4. Peak fractions
were pooled and dialyzed into 150 mM NaCl, 20 mM HEPES, pH 7.8, for cross-linking reactions. SulfoEGS,
SulfoDST, and BS3 (Pierce) were made up as 5 mg/ml stock
solutions and diluted to various protein:cross-linker ratios. H3
protein concentrations ranged from 20 to 100 µM. Reaction
volumes were 30 µl for each cross-linker concentration tested, and
cross-linking reactions were carried out at 25 °C for 30 min.
Reactions were quenched by adding an equal volume of buffer containing
1% sodium dodecyl sulfate (SDS), 15% glycerol, 15 mM
dithiothreitol, 0.2% Coomassie Blue dye and 125 mM Tris,
pH 6.9, and then boiled for 5 min. Samples were then subjected to
SDS-PAGE using 15% acrylamide.
Gel Filtration--
To determine the oligomeric state of the
wild type and mutant F216A and F216L syntaxin 1a H3 proteins, fractions
containing ~95% pure protein (as determined by SDS-PAGE, data not
shown) were applied to a Superdex 200 HR10/30 or Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech) in a buffer containing 150 mM NaCl, 2 mM Circular Dichroism Measurements--
Circular dichroism (CD)
spectroscopy and melting temperature experiments were performed
on fast protein liquid chromatography-purified protein dialyzed against
a buffer containing 150 mM NaCl and 20 mM Tris,
pH 8.0, or 150 mM NaCl and 20 mM sodium
acetate, pH 5.5. The protein concentration for all samples was 126 µM (1.1 mg/ml). Data were collected using an AVIV 62 A DS
CD spectrometer. The higher molar ellipticity values for some of the
samples result from differences in helical content and not differences
in protein concentration. To determine thermal stabilities
(Tm values), Structure Determination--
The H3 region of rat syntaxin 1a,
consisting of residues 191-267, was expressed in E. coli as
a glutathione S-transferase (GST) fusion protein and
purified after thrombin cleavage of the GST tag. Crystals were obtained
in the pH range 5.0-5.3 from a precipitant containing a mixture of
tartrate and acetate salts. Most crystals grew in a trigonal space
group that contained two molecules in the asymmetric unit, which
typically diffracted to 3.2 Å. The final model, discussed below, was
refined against a native data set to 2.4-Å resolution obtained from a
related but nonreproducible crystal form that contains four copies of
the H3 peptide in the asymmetric unit. Data collection, phasing,
and refinement statistics are given in Tables
I, II, and
III.
Structure of the H3 Region of Syntaxin 1a and Comparison with the
Core SNARE Complex Structure--
Each of the four copies of H3 in the
asymmetric unit of the crystal forms an extended, amphipathic
There are slight conformational differences among the four helices in
the H3 tetramer. Copies A and B are parallel, as are C and D. Copies A
and C are more ordered at their N and C termini, with electron density
visible for residues 190-256 and 190-255, respectively. In addition,
copy C contains an interruption in the
The fact that the H3 region forms an extended helical assembly makes it
of interest to compare the H3 homotetramer structure with the core
SNARE complex structure (7). The most obvious difference between these
two structures is that all of the long helices are parallel to each
other in the core SNARE complex, whereas the H3 tetramer consists of
anti-parallel pairs of parallel helices. Another difference is that the
H3 region in the core complex structure is structured further toward
the C terminus. The last ordered syntaxin 1a residue seen in the
crystal structure of the core SNARE complex is 259 or 261 (seen in
different copies in the asymmetric unit), whereas in the H3 tetramer
the last visible residue is either 253 or 256. Both crystal structures
show more order at the C terminus than NMR solution studies of
full-length syntaxin like proteins (11, 24), in which the H3 region is disordered from residues 226-267. This discrepancy may arise from the
fact that in full-length syntaxin, the interaction with Habc stabilizes
the first half of the H3 peptide, leaving the rest disordered.
Alternatively, the high local concentration of the H3 peptide in the
crystal, the nature of the crystallization conditions, or preferential
incorporation of the more helical population into the crystal lattice
may make the present structure more ordered than the ensemble present
in solution. Electron density maps calculated from data measured from
crystals diffracting to a lower resolution reveal that the C terminus
of each copy of H3 is less well ordered from residues 247 to 256 than
in the structure presented here, again suggesting that these residues
are ordered only under limited conditions.
Copy A from the H3 tetramer superimposes on VAMP from the core SNARE
complex with an r.m.s.d. of 2.3Å, and copy B from the H3 tetramer
superimposes on H3 from the core SNARE complex with an r.m.s.d. of
0.4Å on C
The core of the H3 four-helix bundle is composed primarily of
hydrophobic residues, and these same residues face the interior of the
core SNARE complex (7). However, each protomer contributes one polar
residue (Gln-226) to the hydrophobic core of the four-helix bundle, and
in each set of parallel helices these glutamine residues are aligned in
the same register and are hydrogen-bonded to one another. The pair of
glutamine 226 residues from parallel helices packs against two
phenylalanine 216 residues contributed by the opposing anti-parallel
helices (Fig. 3A). In the core
SNARE complex structure Gln-226 forms part of a central polar layer,
interacting with Arg-56 of VAMP, and Gln-53 and Gln-174 of SNAP25 (Fig.
3B). Phe-216 forms part of a hydrophobic layer, where it is
packed against small residues present in SNAP-25 and VAMP2 (Fig.
3C).
Several noteworthy features are present on the surface of the H3
tetramer. The H3 peptide is slightly acidic (calculated pI = 6.1),
and displays a large area of electrostatically negative surface
potential (Fig. 4). The H3 tetramer
contains only two small regions of positive charge in the central part
of the structure, attributable to Arg-246 from copy A and Arg-232 from
copy B. Interestingly, both the H3 tetramer and the core SNARE complex
have predominantly electrostatically negative surface potentials, and
both form long narrow structures (Fig. 4). In addition, both bind
Inspection of the H3 tetramer surface also reveals a potential docking
site for a small molecule. A tartrate ion is bound in a pocket formed
between Gly-227 of copy A and His-212 of copy C. Interestingly, this is
the site of the helix interruption in copy C. This structure cannot be
attributed to lattice interactions, because the site is well away from
any neighbors in the crystal lattice. Also, the mercury compound
(2,6-bis-chloromercuri-4-nitrophenol), used as the heavy atom
derivative, binds in this site. The mercurial is covalently bound to a
disordered part of the polypeptide chain of a neighboring molecule in
the crystal lattice but was found ordered in the Gly-227/His-212
surface cavity. The cavity seems to be somewhat selective, because
several other mercurial compounds that could be covalently attached to
Cys-257 solution crystallized but did not show any ordered mercury atoms.
H3 Forms Oligomers in Solution--
To assess the significance of
the crystallographically observed H3 tetramer, the oligomeric state of
H3 in solution was investigated by gel filtration and chemical
cross-linking. The predicted molecular mass for an H3 tetramer is ~40
kDa; however, the H3 peptide runs at an apparent molecular mass of 60 kDa on a gel filtration column (Fig.
5A). The crystal structure
reveals that the H3 tetramer is a long and narrow assembly, so the high
molecular weight estimate obtained from the sizing column may be due in
part to the elongated shape of the oligomer. Experiments using three
different homobifunctional lysine-reactive cross-linking agents also
support the existence of H3 oligomers in solution (data for
BS3 in Fig. 6A;
others not shown). At low concentrations of cross-linker, dimeric
species predominate. As the concentration of Sulfo-EGS (16.1-Å spacer, data not shown) or BS3 (11.4-Å spacer) is raised, the
molecular mass of the predominant species as estimated by SDS-PAGE is
~28 kDa, which is consistent with a trimer. Only small amounts of tetramer can be detected. The cross-linker with the shortest spacer arm, Sulfo-DST (6.4 Å), produced only dimeric species even at high
ratios of cross-linker to protein (data not shown).
The crystal structure of the tetramer is consistent with the observed
cross-linking patterns. The three ordered lysine residues in one copy
of the H3 polypeptide are at position 204 near the N-terminal end and
positions 252 and 253 near the C-terminal end (Fig. 7). The dimers are
likely a result of cross-linking of pairs of lysines from parallel
helices. For example, the terminal nitrogen atoms of lysines 204 from
copies C and D are 13.8 Å apart in the crystal structure, and the
terminal nitrogen atoms of lysines 252 and 253 from adjacent parallel
helices A and B are 10.1 Å apart in the structure. Modeling indicates
that small changes in rotamers would place the 252/253 pair within
cross-linking range of even the shortest cross-linker used in these
experiments, Sulfo-DST. In contrast, the trimeric species formed with
the longer cross-linking agents are likely due to reactions between
Lys-204, Lys-252, and Lys-253 (Fig. 7).
At each end of the H3 tetramer, Lys-252 and Lys-253 from parallel
helices are within cross-linking distance of each other, but only one
of the Lys-204 side chains from the anti-parallel helices is in close
enough proximity to form cross-links to either of the Lys-252 or
Lys-253 side chains.
The stability of the H3 oligomer was assessed by using circular
dichroism (CD) spectroscopy to measure melting temperature (Tm) transitions. Data were measured both at pH
8.0 and 5.5, to reflect the purification and crystallization
conditions, respectively; pH 5.5 was as close to the crystallization pH
as was obtainable without causing the protein to precipitate.
Consistent with previous studies (25, 26), the CD spectra indicate that
the H3 polypeptide is mostly helical. The average
Tm for the wild type H3 oligomer is 47 °C at
pH 8.0 and 58 °C at pH 5.5 (Fig.
8A). It has been noted that
acidic conditions can induce or stabilize Determinants of Bundle Topology and Stability--
Polar residues
are buried in the core of several parallel helix assemblies, including
the core SNARE complex tetramer, GCN4 homodimers (28, 29), Jun
homodimers and other leucine zipper proteins (30), and the influenza
virus hemagglutinin (31). Given the all-parallel topology of the core
SNARE complex and the precedence for polar layers in the hydrophobic
cores of oligomeric bundle structures, we investigated why the H3
fragment does not favor an all-parallel arrangement. In the H3
tetramer, two Gln-226 residues form hydrogen bonds with each other and
are packed against two Phe-216 residues present on the anti-parallel
dimer pair (Fig. 3A). Modeling suggests that four glutamine
residues could form a mutually interacting set of side chains in an
all-parallel arrangement of four H3 helices. However, this topology
would require that all four Phe-216 residues pack against one another
in the core, and it appears that this cannot occur without introducing
significant distortions into the backbone. In the core SNARE complex,
Phe-216 of syntaxin 1a packs against several smaller side chains:
Gly-43 and Ala-164 from SNAP-25, and Val-42 from VAMP2 (Fig.
3C). Sequence alignment of the H3 region of several
different syntaxin-like proteins reveals that the phenylalanine and
glutamine residues, as well as their positions relative to each other,
are highly conserved (32, 33). It has been suggested that the
complementarity of the large Phe-216 with smaller residues helps to
ensure the correct register of helices in the core complex (33). The
anti-parallel H3 tetramer suggests that Phe-216 may also prevent
formation of stable but unproductive parallel syntaxin tetramers, thus
allowing the H3 region to bind other proteins such as VAMP and
SNAP-25.
In the absence of the large Phe-216 side chain, it is possible that all
four H3 helices might have a parallel orientation. To test this
hypothesis, H3 mutants Phe-216
The stabilities of the F216L and F216A mutants were measured at pH 8.0 and 5.5 and compared with the wild type protein. At pH 8.0, the F216L
mutant has a higher melting temperature than wild type H3 (56 °C
versus 47 °C, Fig. 8B). At pH 5.5, the melting curve of F216L is biphasic, with Tm values of
79 °C and 18 °C (Fig. 8B). If this represents two
distinct populations consisting of anti-parallel and parallel tetramers
with low and high Tm values, respectively, then
there is likely a kinetic barrier to their interconversion. To test
this, the protein was brought to 45 °C and then slowly cooled to
4 °C (see "Experimental Procedures"). This protocol should
"anneal" the helical bundles and allow formation of only the more
stable population. Temperature melting data obtained from annealed
samples shows only one melting transition at 83 °C instead of two
(Fig. 8C). This evidence supports the hypothesis that the
F216L mutation allows the H3 peptide to form anti-parallel and parallel
tetramers, and that the parallel species is more stable.
The F216A protein was very unstable as judged by CD temperature melting
profiles at pH 8.0, where the peptide seems to have little
Collectively, these results suggest that Phe-216 of the wild type
sequence sterically prevents parallel tetramer formation and that the
presence of Leu at position 216 allows parallel tetramer formation. The
more facile cross-linking properties and the biphasic melting curve of
the F216L mutant may reflect a mixed population of all-parallel and
anti-parallel tetramers, with the more stable melting phase
representing the all-parallel arrangement. Surprisingly, the presence
of Ala at position 216 prevents formation of extensive secondary
structure at pH 8.0. Presumably, the tetrameric state and enhanced
stability of F216A at acid pH reflects compensatory stabilization by
hydrogen bonds, as discussed above for the wild type protein.
The H3 region of syntaxin 1a can form binary complexes with its
partner t-SNARE SNAP-25, and this interaction may be an obligatory intermediate in the formation of the SNARE complex (4, 12). In
vitro, syntaxin 1a and SNAP-25 interact to form a binary complex that significantly enhances their affinity for VAMP over either component alone (2, 24, 34), and this interaction is the rate-determining step in forming the full SNARE heterotrimer (12). The
stoichiometry of the binary complex is unclear, but EPR experiments indicate that the complex is a parallel four-helix
bundle.4 CD data obtained
using Sso1p and Sec9p (the yeast syntaxin 1a and SNAP-25 homologs)
indicate that these proteins form a 1:1 complex, whereas data obtained
from the neuronal syntaxin 1a and SNAP-25 proteins support the presence
of a 1:1 or 2:1 complex (2).5
Data obtained from yeast and neuronal systems support the participation of SNAP-25 N-terminal regions in the binary complex (4, 12), but it is
unclear if the C-terminal regions are also involved.
Assuming that the SNAP-25·syntaxin 1a binary complex forms a
four-helix bundle structure, it is possible that the parallel syntaxin
helices observed in the H3 tetramer structure represent two copies of
syntaxin 1a in a complex with SNAP-25. The preformed syntaxin dimer may
assist SNAP-25 in adopting Analytical ultracentrifugation analysis has revealed that the
full-length syntaxin cytoplasmic domain forms dimers and a small amount
of tetramer in solution (6). This oligomerization was attributed to the
H3 region. It seems likely that the assembly observed in the H3 crystal
structure represents the behavior of H3 alone as well as the assembly
of full-length syntaxin 1a. The H3 construct used here extends to
residue 267, but electron density is not seen for residues beyond 253. This observation is curious, because syntaxin 1-265 oligomers appear
to be more stable than those of syntaxin 1-253 (6). The dimerization
constant for syntaxin 1-265 is 5.8 µM, and the
dimer-tetramer constant is 12 µM (6). The CD and
cross-linking experiments reported by Lerman et al. (6) were
performed at high concentrations (>75 µM), similar to
those used in the experiments reported here. Therefore, we might have
expected to see some dimers in the H3 gel filtration experiments.
However, only one peak was observed, suggesting that the H3 region may
oligomerize more easily in the absence of the Habc domain. This may be
due to the competing interaction of H3 with the Habc region in the
full-length cytoplasmic domain of syntaxin.
In v-SNARE-deleted yeast strains a low level of homotypic vacuole
fusion is observed (35), suggesting that complexes of syntaxin
tetramers might mediate homotypic fusion (6). The results presented
here, however, suggest that syntaxin-like proteins are unable to form
all-parallel homotetramers. Therefore, if some level of homotypic
fusion is mediated by syntaxin-like proteins alone, this mechanism of
fusion is different from syntaxin·VAMP·SNAP-25-mediated fusion
involving parallel four-helix bundles. It is possible that anti-parallel syntaxin tetramers could form between molecules anchored
in opposing membranes, and such an arrangement would bring the
membranes close enough to allow fusion to occur. However, data
presented here indicate that such structures would be less stable than
all-parallel SNARE complex assemblies.
The highly conserved Phe-216 residue seems to prevent formation of
stable parallel H3 tetramers. This may be important to ensure that
productive complexes, such as the binary SNAP-25 complex or the core
SNARE complex, can be formed readily without competition from a
self-association reaction. It is likely that substitution of leucine
for phenylalanine at position 216 allows parallel four-helix bundle
assembly due to relief of steric constraints imposed by the large
phenylalanine side chain. It is also possible that the Phe-216
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside. Cells were
harvested by centrifugation and lysed by French press in the presence
of phenylmethylsulfonyl fluoride. The lysate was mixed with
glutathione-agarose beads (2 ml of 50% beads for each liter of cells)
and incubated for 1 h at 4 °C. The beads were washed with
buffer containing 250 mM NaCl, 2 mM
CaCl2, 2 mM
-mercaptoethanol (
-Me), 50 mM Tris, pH 8.0, and the fusion protein was cleaved with
Thrombin (Sigma Chemical Co.). The supernatant was applied to a Mono Q HR10/30 (Amersham Pharmacia Biotech) column at 4 °C (10 mM or 1 M NaCl, 2 mM
-Me, and 15 mM Tris, pH 8.4). For crystallization, the Mono Q fractions
were concentrated to ~15 mg/ml using Centriprep concentrators with a
molecular mass cutoff of 3 kDa (Amicon).
radiation from a rotating anode
(Rigaku; 50 kV, 90 mA, graphite monochromator, 0.3-mm collimator). MAD
data were measured at four wavelengths at beamline 5.02 of the Advanced
Light Source, using a Quantum4 charge-coupled device detector
(Area Detector Systems Corp.). Friedel pairs were systematically
measured using inverse beam geometry. All data were collected at 100 K
and processed and scaled with Denzo/Scalepack (17). Data collection
statistics are presented in Table I below.
3)-positive Friedel data used as a reference. The phases were
improved using solvent flattening and histogram matching as implemented
in the program DM (19), with an estimated solvent content of 60%. The
improved phases and increased resolution produced an interpretable
electron density map. The asymmetric unit contains two parallel
helices, which associate with a crystallographic 2-fold
symmetry-related pair to form the four-helix bundle. Phasing statistics
are presented in Table II below.
A-weighted (2Fo
Fc) maps, conjugate gradient minimization, and
individual thermal factor refinement were then carried out using the
native data set to produce the final model. Several strong water
molecules were identifiable, and additional water molecules were placed in areas that contained positive difference Fourier peaks greater than
3
and that exhibited a sensible chemical environment. The final
model contains residues 190-256 for copy A, 193-254 for copy B,
190-255 for copy C, 193-253 for copy D, 53 water molecules, and one
tartaric acid molecule. Refinement statistics are presented in Table
III below.
-Me, and 20 mM
Tris, pH 8.0, or 20 mM sodium acetate, pH 5.5. The peak
elution volume was compared with protein standards of known molecular
weight (Sigma).
-helical signals were
monitored by the ellipticity at 222 nm while the sample was heated from
4 °C to 90 °C. All apparent Tm values were taken as the maximum of the first derivative of the melting curve. Wavelength spectra and temperature melt data were acquired using a 1-mm
path length quartz cuvette. For thermal unfolding experiments, a 1°
step size and 30-s equilibration time were used. The F216L mutant was
annealed by raising the temperature of the protein sample to 45 °C
in 10° increments and equilibrating for 10 min at each temperature.
The sample was then cooled by lowering the temperature to 4 °C in
5° increments, with a 5-min equilibration time at each temperature.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Data collection statistics
Phasing statistics
Refinement statistics
helix
along the majority of the polypeptide chain (Fig.
1). A parallel dimer formed by two
helices associates in an anti-parallel orientation with another parallel dimer. The hydrophobic faces of the helices form both the
parallel and anti-parallel interfaces. This packing arrangement produces a four-helix bundle with approximate dimensions 100 × 35 × 35 Å (Fig. 1). The topology of the bundle is unusual,
because other anti-parallel four-helix bundles are arranged with
adjacent helices anti-parallel to one another, with the two parallel
helices located across the diagonal (23). The topology observed in the H3 tetramer represents yet another mode by which helices can associate into a tetrameric assembly.
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Fig. 1.
Structure of the anti-parallel H3 four-helix
bundle. N- and C termini are indicated, along with the
numbers of ordered residues for each copy. The Phe-216 and Gln-226 side
chains face the center of the bundle and are drawn as
balls and sticks. This figure, Fig. 3, and Fig. 7 were made
with MOLSCRIPT (36) and rendered with RASTER3D (37).
-helical backbone at residue
212. Copies B and D are shorter (residues 196-254 and 194-253,
respectively). The N-terminal ends of copies B and D are significantly
straighter than their C-terminal ends, and as a result the ends of the
four-helix bundle are not as tightly assembled as the central region.
positions (Fig.
2A). Either set of two parallel helices in the H3 tetramer does not superimpose with any two
helices in the core SNARE complex structure. This is likely due to
differences in packing angles between helices, where the values are
between 15° and 20° for the H3 tetramer and between 5° and 20°
for the core complex structure. The H3 parallel dimers also do not have
as much of a left-handed superhelical twist as the dimers in the core
SNARE complex structure (Figs. 2C and 4).
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Fig. 2.
Superpositions of the H3 tetramer and the
core SNARE complex. Helices from the H3 tetramer are shown in
gray, helices from the core SNARE complex structure are
shown in yellow (VAMP) and purple (syntaxin 1a
H3). A, superposition of copy A from the H3 tetramer on
VAMP2 from the core SNARE complex structure (2.3-Å r.m.s.d.). VAMP2
residues 30-92 from the core SNARE complex structure were rotated onto
H3 tetramer residues 194-253 from H3 copy A. B,
superposition of copy B from the H3 tetramer on syntaxin 1a H3
from the core SNARE complex structure (0.4Å r.m.s.d.). Syntaxin 1a H3
residues 194-253 from the core SNARE complex structure were rotated
onto H3 tetramer residues 194-253 from copy B. C, copies A
and B from the H3 tetramer do not superimpose with VAMP and syntaxin 1a
H3 from the core complex (8.2Å r.m.s.d.). The VAMP2 and H3 helices
from the core SNARE complex were rotated together onto two helices from
the H3 tetramer to give the best fit. The residues included in this
superposition were the same as for panels A and
B. This figure and Fig. 4 were made using the program GRASP
(18).
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Fig. 3.
Layers in the H3 and core SNARE complex
four-helix bundle structures viewed parallel to the helical axis.
A, one of the Phe-216/Gln-226 layers from the H3 structure,
colors are the same as for Fig. 1. B, the ionic
layer in the core SNARE complex structure (7), which includes Gln-226
from syntaxin 1a H3. C, a layer from the core SNARE complex
structure showing the interaction of Phe-216 with side chains from the
other SNARE proteins. VAMP2 is shown in yellow, syntaxin 1a
H3 is shown in purple, and SNAP-25 in
orange.
-SNAP and NSF to form a 20 S
particle3 (Ref. 15 and
references therein).
-SNAP and NSF are general factors that function
in multiple transport pathways and are thus capable of associating with
many different SNARE proteins.
-SNAP and NSF may recognize SNARE
protein assemblies based on surface complementarity and electrostatic
surface potential rather than specific sequences, thus accounting for
the versatility of these proteins.
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Fig. 4.
Comparison of the electrostatic surface
potential and structure of the SNARE core complex and H3 tetramer.
A, electrostatic surfaces of the core complex and H3
tetramer are drawn as a mesh frame over ribbon
diagrams of each structure, the syntaxin 1a H3 peptide is shown in
black. B, ribbon diagrams of the core
SNARE complex and H3 tetramer are shown in the same orientation as in
A.
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Fig. 5.
Summary of sizing column data at pH 8.0 and
5.5. A, apparent molecular weights for wild type,
F216A, and F216L H3 peptides when eluted from a Superdex 200 gel
filtration column at pH 8.0. B, apparent molecular weights
for wild type, F216A, and F216L H3 peptides when eluted from a Superdex
200 gel filtration column at pH 5.5. C, the shift of the
elution peak of the F216A mutant under acidic conditions (pH 5.5)
relative to basic conditions (pH 8.0) is shown. The elution profile of
wild type H3 protein at pH 8.0 is shown for comparison.
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Fig. 6.
Representative cross-linking results as
analyzed by SDS-PAGE. Only results obtained with the
BS3 cross-linker (11.4-Å spacer arm) are shown, and
estimated molecular weights are indicated. For each protein the lanes
from left to right are: control, no
cross-linker added, 66 µM protein (0.56 mg/ml);
1, 15:1 ratio protein:cross-linker; 2, 5:1 ratio
protein:cross-linker; 3, 1:2 ratio protein:cross-linker;
4, 1:5 ratio protein:cross-linker; 5, 1:10 ratio
protein:cross-linker; 6, 1:20 ratio protein:cross-linker;
7, 1:40 ratio protein:cross-linker; 8, 1:60 ratio
protein:cross-linker; and 10-kDa ladder markers are shown in the
far right lane. A, wild type protein;
B, F216L protein; C, F216A protein. Note that the
monomeric H3 peptide runs at 8 kDa rather than the expected 10 kDa.
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Fig. 7.
Enlarged view of one end of the H3
tetramer. Lysine side chains are drawn as balls and
sticks, and distances between selected pairs are shown to indicate
likely cross-linking sites.
helical structure by
stabilizing the hydrogen bond network between acidic side chains (27).
The enhanced stability at pH 5.5 may be due to the formation of salt
bridges between glutamate residues adjacent to one another on the
outside of the helix (Glu-224 and Glu-228, and Glu-234 and
Glu-238).
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Fig. 8.
Representative wavelength scans for the wild
type, F216A, and F216L proteins at 25 °C and pH 8.0 (A) or pH 5.5 (B). Data measured
at pH 8.0 are shown as filled circles, and data collected at
pH 5.5 are shown as open circles. Red,
green, and purple curves represent data from the
wild type, F216A mutant, and F216L mutant proteins, respectively.
C, D, and E show representative
temperature melting curves for wild type, F216A, and F216L H3 proteins,
respectively. For the temperature melting experiments, the helical
signal was monitored at 222 nm as the temperature was varied from
10 °C to 90 °C. The black curve in E shows
data measured from the F216L mutant protein at pH 5.5 after annealing
(see text).
Ala (F216A) and Phe-216
Leu
(F216L) were prepared. Attempts to crystallize the mutant proteins
failed, suggesting that the mutant proteins failed to form the same
oligomeric complexes as the wild type protein, they are significantly
less stable than the wild type protein, or they have an otherwise
significantly altered structure that prevents formation of the same
lattice contacts as the wild type protein. In the absence of direct
structural data, it is unclear if these mutations relieve the steric
constraint provided by the phenylalanine residues and allow a parallel
tetramer to form. We therefore examined the oligomeric states of the
mutant proteins and stabilities relative to the wild type protein. Gel
filtration at pH 8.0 reveals that the F216L mutant protein runs at the
same apparent molecular weight as the wild type protein (Fig.
5A). Unlike the wild type protein, however, F216L can be
readily cross-linked to tetramers, and dimeric species are still
present at the highest ratio of cross-linker:protein (Fig.
6B). In contrast, the F216A mutant protein runs at a lower
molecular mass (~32 kDa) on a gel filtration column at pH 8.0 (Fig.
5A). When this mutant is exposed to either SulfoEGS or
BS3 cross-linking agents (Fig. 6C), a dimeric species is observed but the protein remains largely monomeric.
helical
secondary structure and the Tm = 30 °C (Fig. 8D). This is consistent with observations that the F216A
protein had a tendency to degrade during purification. Surprisingly,
acidic conditions had a more dramatic effect on the stability of the F216A protein in comparison to the wild type or F216L mutant proteins; where the Tm was 48 °C (Fig. 8D).
Given this result, the gel filtration profiles for the three proteins
were examined at pH 5.5 (Fig. 5B). The elution profiles of
the wild type and F216L protein remained at the presumed tetramer
position seen at pH 8.0, but the F126A protein clearly shifted from the
~32-kDa position to the ~60-kDa position (Fig. 5, B and
C). This indicates that the F216A protein forms tetramers at
pH 5.5, and this change in oligomeric state may explain the enhanced
stability of the F216A mutant under acidic conditions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical structure, thus providing a
binding site for VAMP and allowing SNARE complex formation. If syntaxin
1a and SNAP-25 are present in a 2:1 molar ratio, then it is likely that
both the N- and C-terminal regions of SNAP-25 bind to the syntaxin
dimer. If the stoichiometry of the two proteins is 1:1 (overall
composition of 2:2), however, then it is likely that only the
N-terminal region of SNAP-25 forms the binary complex with syntaxin 1a
H3. Molecular modeling indicates no obvious steric clashes to prevent
formation of either type of parallel binary SNAP-25·H3 complexes, but
further biophysical and structural characterization will be required to
determine the nature and relevance of the syntaxin 1a·SNAP-25 complex.
Ala
mutant protein forms parallel four-helix bundles under acidic
conditions. The thermal stability of this mutant protein
(Tm = 48 °C) is lower than expected for a
very stable parallel four-helix bundle such as the SNARE complex
(Tm = 90 °C), but this may be due in part to
suboptimal packing of the hydrophobic core caused by the small alanine
side chain. The effect of Phe-216 mutations on the assembly of syntaxin
H3 demonstrates that steric complementarity has important
effects on the assembly of helical oligomers. The conservation of
Phe-216 may be important both in insuring correct register of SNARE
helices (33), and also to prevent unproductive modes of syntaxin
self-association.
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ACKNOWLEDGEMENTS |
---|
We thank K. Ervin, R. Hollomon, and T. Hong for technical assistance; T. Earnest and the staff at the Advanced Light Source for beamline support; M. Soltis and H. Bellamy at the Stanford Synchrotron Radiation Laboratory for beamline support and advice with heavy atom derivatization; A. Kolatkar for assistance with data collection; and A. Kolatkar, L. Gonzalez, R. Samudrala, A. May, and J. Wedekind for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants MH58570 (to W. I. W.) and MH38710 (to R. H. S.) from the National Institute of Mental Health. Part of this work is based upon research conducted at the Stanford Synchrotron Radiation Laboratory, which is funded by the Department of Energy, Offices of Basic Energy Sciences, and Biological and Environmental Research; the National Center for Research Resources, Biomedical Technology Program, NIH; and the National Institute of General Medical Sciences. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Science Division, of the U. S. Department of Energy under contract DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.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.
¶ Supported by Molecular Biophysics Training Grant T32-GM08294 from NIH.
** To whom correspondence should be addressed: Dept. of Structural Biology, Stanford University School of Medicine, 299 Campus Dr. West, Stanford, CA 94305. Tel.: 650-725-4623; Fax: 650-723-8464; E-mail: bill.weis@stanford.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M009636200
2 T. C. Terwilliger and J. Berendzen, Los Alamos National Laboratory; available on the Web.
3 A. May, personal communication.
4 M. Poirier, personal communication.
5 L. Gonzalez, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
NSF, N-ethylmaleimide-sensitive factor;
SNARE, SNAP
receptor;
v-SNARE, vesicle SNARE;
t-SNARE, target SNARE;
-Me,
-mercaptoethanol;
MAD, multiwavelength anomalous dispersion;
PAGE, polyacrylamide gel electrophoresis;
CD, circular dichroism;
GST, glutathione S-transferase;
r.m.s.d., root mean square
deviation;
SNAP, soluble NSF attachment protein.
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REFERENCES |
---|
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---|
1. | Jahn, R., and Sudhof, T. C. (1999) Annu. Rev. Biochem. 68, 3250-3262 |
2. |
Fasshauer, D.,
Otto, H.,
Eliason, W. K.,
Jahn, R.,
and Brunger, A. T.
(1997)
J. Biol. Chem.
272,
28036-28041 |
3. | Hazzard, J., Sudhof, T. C., and Rizo, J. (1999) J. Biomol. NMR 14, 203-207[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Fasshauer, D.,
Bruns, D.,
Shen, B.,
Jahn, R.,
and Brünger, A. T.
(1997)
J. Biol. Chem.
272,
4582-4590 |
5. | Fernandez, I., Ubach, J., Dulubova, I., Zhang, X., Sudhof, T. C., and Rizo, J. (1998) Cell 94, 841-849[Medline] [Order article via Infotrieve] |
6. | Lerman, J. C., Robblee, J., Fairman, R., and Hughson, F. M. (2000) Biochemistry 39, 8470-8479[CrossRef][Medline] [Order article via Infotrieve] |
7. | Sutton, R. B., Fasshauer, D., Jahn, R., and Brünger, A. T. (1998) Nature 395, 347-353[CrossRef][Medline] [Order article via Infotrieve] |
8. | Misura, K. M. S., Scheller, R. H., and Weis, W. I. (2000) Nature 404, 355-362[CrossRef][Medline] [Order article via Infotrieve] |
9. | Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Südhof, T. C., and Niemann, H. (1994) EMBO J. 13, 5051-5061[Abstract] |
10. | Rice, L. M., Brennwald, P., and Brunger, A. T. (1997) FEBS Lett. 415, 49-55[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Dulubova, I.,
Sugita, S.,
Hill, S.,
Hosaka, M.,
Fernendez, I.,
Sudhof, T. C.,
and Rizo, J. A.
(1999)
EMBO J.
18,
4372-4382 |
12. | Nicholson, K. L., Munson, M., Miller, R. B., Filip, T. J., Fairman, R., and Hughson, F. M. (1998) Nature Struct. Biol. 5, 793-802[CrossRef][Medline] [Order article via Infotrieve] |
13. | Fujita, Y., Shirataki, H., Sakisaka, T., Asakura, T., Ohya, T., Kotani, H., Yokoyama, S., Nishioka, H., Matsuura, Y., Mizoguchi, A., Scheller, R. H., and Takai, Y. (1998) Neuron 20, 905-915[Medline] [Order article via Infotrieve] |
14. |
Rettig, J.,
Sheng, Z.-H.,
Kim, D. K.,
Hodson, C. D.,
Snutch, T. P.,
and Catterall, W. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7363-7368 |
15. | Woodman, P. G. (1997) Biochim. Biophys. Acta 1357, 155-172[Medline] [Order article via Infotrieve] |
16. | Kee, Y., and Scheller, R. H. (1996) J. Neurosci. 16, 1975-1981[Abstract] |
17. | Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326 |
18. | Nicholls, A. (1992) GRASP: Graphical Representation and Analysis of Surface Properties , Columbia University, New York |
19. | Cowtan, K. D., and Main, P. (1996) Acta Cryst. D52, 43-48 |
20. | Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
21. | Brünger, A. T., Adams, P. D., Clore, G. M., 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. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
22. | Pannu, N. S., and Read, R. J. (1996) Acta Crystallogr. Sect. A 52, 659-668[CrossRef] |
23. | Murzin, A. G., Lo Conte, L., Ailey, B. G., Brenner, S. E., Hubbard, T. J. P., and Chothia, C. (1995) J. Mol. Biol. 247, 536-540[CrossRef][Medline] [Order article via Infotrieve] |
24. | Fiebig, K. M., Rice, L. M., Pollock, E., and Brunger, A. T. (1999) Nature Struct. Biol. 6, 117-123[CrossRef][Medline] [Order article via Infotrieve] |
25. | Fasshauer, D., Eliason, W. K., Brunger, A. T., and Jahn, R. (1998) Biochemistry 37, 10354-10362[CrossRef][Medline] [Order article via Infotrieve] |
26. | Zhong, P., Chen, Y. A., Tam, D., Chung, D., Scheller, R. H., and Miljanich, G. P. (1997) Biochemistry 36, 4317-4326[CrossRef][Medline] [Order article via Infotrieve] |
27. | Zhou, N. E., Kay, C. M., and Hodges, R. S. (1994) J. Mol. Biol. 237, 500-512[CrossRef][Medline] [Order article via Infotrieve] |
28. | O'Shea, E. K., Klemm, J. D., Kim, P. S., and Alber, T. (1991) Science 254, 539-544[Medline] [Order article via Infotrieve] |
29. | Harbury, P. B., Zhang, T., Kim, P. S., and Alber, T. (1993) Science 262, 1401-1407[Medline] [Order article via Infotrieve] |
30. | Junius, F. K., Mackay, J. P., Bubb, W. A., Jensen, S. A., Weiss, A. S., and King, G. F. (1995) Biochemistry 34, 6164-6174[Medline] [Order article via Infotrieve] |
31. | Wilson, I. A., Skehel, J. J., and Wiley, D. C. (1981) Nature 289, 366-373[Medline] [Order article via Infotrieve] |
32. | Weimbs, T., Mostov, K. E., Low, S. H., and Hofmann, K. A. (1998) Trends Cell Biol. 8, 260-262[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Fasshauer, D.,
Sutton, R. B.,
Brunger, A. T.,
and Jahn, R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15781-15786 |
34. | Pevsner, J., Hsu, S.-C., Braun, J. E. A., Calakos, N., Ting, A. E., Bennett, M. K., and Scheller, R. H. (1994) Neuron 13, 353-361[Medline] [Order article via Infotrieve] |
35. | Nichols, B. J., Ungermann, C., Pelham, H. R. B., Wickner, W. T., and Haas, A. (1997) Nature 387, 199-202[CrossRef][Medline] [Order article via Infotrieve] |
36. | Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
37. | Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524 |