(Received for publication, September 8, 1996, and in revised form, November 13, 1996)
From the § Howard Hughes Medical Institute and the
The highly conserved proteins syntaxin and
SNAP-25 are part of a protein complex that is thought to play a key
role in exocytosis of synaptic vesicles. Previous work demonstrated
that syntaxin and SNAP-25 bind to each other with high affinity and
that their binding regions are predicted to form coiled coils. Circular
dichroism spectroscopy was used here to study the Neurotransmitters are released from presynaptic nerve endings by
Ca2+-triggered exocytosis of synaptic vesicles. Several
lines of evidence suggest that exocytotic membrane fusion is mediated
by a complex of conserved proteins which includes the synaptic vesicle
protein synaptobrevin (also referred to as vesicle-associated membrane protein or VAMP) and the synaptic membrane proteins syntaxin and SNAP-25. Homologs of these neuronal proteins have been identified in
many non-neuronal cell types including the yeast Saccharomyces cerevisiae, suggesting that the mechanism of exocytotic membrane fusion is conserved in all eukaryotic cells (for review, see Refs. 1-4).
Although the evidence linking these proteins to membrane fusion is
quite compelling, very little is known about their mechanism of action.
Rothman and colleagues (5) found that the three membrane proteins form
a complex that interacts with additional soluble proteins known to
support membrane fusion in cell-free extracts (5). These soluble
proteins include the SNAPs1 (soluble NSF
attachment proteins) with three isoforms ( Ultimately, vesicle docking and membrane fusion can be viewed as a
series of sequential protein assembly and disassembly steps which may
involve structural changes and regulated interactions of at least some
of the proteins with the participating phospholipid bilayers. It is
therefore of interest to understand the structural basis of these
processes.
The three proteins synaptobrevin, syntaxin, and SNAP-25 assemble
spontaneously into a complex that sediments at 7 S (5) and which is
resistant to mild treatment by SDS (9, 10). A complex with very similar
properties can be assembled in vitro from recombinant
proteins that lack their transmembrane domains (syntaxin,
synaptobrevin) or lack their posttranslationally added palmitoyl side
chains (SNAP-25), respectively. The in vitro complex is also
resistant to mild SDS treatment and can be disassembled by NSF in the
presence of ATP (9). Each of these three proteins can bind to one of
its two partners, forming binary complexes. SNAP-25 binds syntaxin with
high affinity (EC50 of about 0.4 µM for
SNAP-25) (11), whereas the binding affinity between syntaxin and
synaptobrevin is weakest (12).
Truncation-, deletion-, and site-directed mutagenesis have revealed the
minimal essential domains of each of the proteins which participates in
the formation of the binary and the ternary complexes (9, 12-14). For
the interaction between SNAP-25 and syntaxin, the
NH2-terminal half of SNAP-25 (amino acids 2-82 of SNAP-25)
and the COOH-terminal domain of syntaxin (amino acids 199-243, also
referred to as the H3 domain (14)) are required (9, 13). The H3 domain
of syntaxin is also sufficient to bind synaptobrevin (12, 14).
Interestingly, the interaction of SNAP-25 with synaptobrevin requires
both the NH2- and COOH-terminal domain of the SNAP-25
molecule (13). The binding of synaptobrevin to either syntaxin or
SNAP-25 requires most of the conserved part of synaptobrevin (amino
acids 27-96, excluding the transmembrane region) (9).
We have used circular dichroism (CD) spectroscopy to study the
secondary structure of syntaxin and SNAP-25 and that of their binary
complex. Although CD spectroscopy is incapable of providing detailed
structural information it can be used to assess the approximate As part of our efforts to develop the Retzius cell of
the leech (H. medicinalis) as a model system for studying
the mechanisms of synaptic transmission (15), all experiments were
based on leech syntaxin and leech SNAP-25 isoforms. cDNA clones
encoding H. medicinalis syntaxin and SNAP-25 were isolated
from a For the generation of full-length SNAP-25 (1-212) the sense and
antisense primers were 5
GST fusion proteins
were purified by affinity chromatography on glutathione-Sepharose beads
essentially as described (13) except that 20 mM Tris, pH
7.4, 150 mM NaCl, 1 mM EDTA, 1 mM
DTT was used as chromatography buffer. Fusion proteins containing His6 tags (expressed in pTrcHisA) were purified by
Ni2+-Sepharose chromatography as described (13). Proteins
were eluted by increasing the imidazole concentration stepwise to 40, 80, 120, or 240 mM (in 20 mM Tris, pH 7.4, 500 mM NaCl). Fractions were analyzed for purity by
SDS-polyacrylamide gel electrophoresis (17) and staining with Coomassie
Blue. Imidazole-containing fractions with recombinant protein were
dialyzed against FPLC-buffer A (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM DTT).
His6-tagged proteins were purified further by anion
exchange chromatography on a Mono Q column using an FPLC system
(Pharmacia Biotech Inc.). After loading, the proteins were eluted with
a linear gradient from 100 to 1,000 mM NaCl, and fractions
containing the purified proteins were pooled. For the purification of
the binary complex of SNAP-25 and syntaxin, both purified proteins were
incubated overnight, dialyzed against FPLC-buffer A, loaded on a Mono Q column, and eluted with a linear gradient from 100 to 1,000 mM NaCl. The peak fractions were pooled, and its
homogeneity was verified by size exclusion chromatography on an
HR-10/30 Superdex 200 column (Pharmacia).
Far UV-CD spectra were obtained by
averaging 5-20 scans with a step size of 0.5 nm on an AVIV model 62DS
CD spectrometer at 25 °C. All measurements were performed in a
Hellma quartz cuvette with a path length of 0.1 or 0.5 cm. All CD
spectra were performed with purified His6-tagged proteins
(pTrcHisA). After purification the proteins were dialyzed against 10 mM phosphate buffer, pH 7.4, containing 100 mM
NaCl (standard conditions) and concentrated by ultrafiltration to final
concentrations of 1-10 mg/ml. Protein concentrations of
SNAP-251-90 fragments and of the purified SNAP-25-syntaxin
complex were calibrated by internally standardized amino acid analysis
following acid hydrolysis (carried out by the W. M. Keck Foundation
Biotechnology Resource Laboratory at Yale University) and
subsequentially determined by measuring absorbance at 280 nm. Protein
concentrations of SNAP-25112-212-fragment, syntaxin1-271, and full-length SNAP-25 were determined by the Coomassie Blue binding method (18). Unless indicated otherwise, all
recordings for single proteins were performed in 10 mM
sodium phosphate buffer, pH 7.4, 100 mM NaCl.
The CD spectra of SNAP-25-syntaxin complexes were recorded after
reaching equilibrium following an overnight incubation at 4 °C in 10 mM phosphate buffer, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, comparable to the conditions used for binding assays with GST fusion proteins. To evaluate changes of the CD spectrum
attributable to complex formation, the theoretical noninteracting spectrum was calculated from the spectra of the individual proteins using the equation [ The fractional Size exclusion chromatography
was performed on an HR-10/30 Superdex 200 column (Pharmacia) in 10 mM sodium phosphate buffer, pH 7.4, containing NaCl
concentrations as indicated at a flow rate of 0.5 ml/min at 25 °C.
The elution profiles were monitored photometrically at 280 nm. 200 µl
of protein solution (10 µM of protein) was loaded.
Globular proteins that were used as molecular mass standards were
loaded at a concentration of 1.0 mg/ml and included alcohol
dehydrogenase, bovine serum albumin, ovalbumin, carbonic anhydrase, and
myoglobulin (molecular mass = 150, 66, 45, 29.5, and 17 kDa,
respectively).
Soluble proteins were incubated together with indicated
amounts of GST fusion protein immobilized on glutathione-Sepharose beads in binding buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 2 mM
MgCl2, 1 mM DTT). Incubations were carried out
overnight at 4 °C. The beads were then washed three times in 1 ml of
binding buffer. Proteins bound to the beads were finally solubilized in SDS sample buffer (final concentrations: 60 mM Tris, pH
6.8, 2% SDS, 10% glycerine, 3% For the prediction of coiled
coil domains the Lupas (20, 21) and the paircoil algorithm (22) were
used. The programs were accessed through the
Internet.3
ulrec3.unil.ch/software/COILS_form.html and
http://ostrich.lcs.mit.edu/cgi-bin/score).
CD spectra of SNAP-25
and syntaxin are shown in Fig. 1A in 10 mM phosphate buffer and 100 mM NaCl, pH 7.4. The CD spectrum of syntaxin, with two clearly defined minima at 208 and
222 nm, suggests an
For more detailed studies of the interaction between SNAP-25 and
syntaxin, a NH2-terminal fragment of SNAP-25 was generated which comprises the minimal domain required for syntaxin binding (SNAP-251-90wt). This fragment showed a slightly higher Increasing NaCl concentrations lead to a dramatic increase
3of the
Divalent cations were about 100-fold more potent than monovalent
cations in inducing
As shown in Fig. 4, lowering the pH to 6.0 also
increased the
As mentioned above, the In the next
series of experiments we investigated whether the binding of SNAP-25 to
syntaxin was associated with changes in secondary structure. For this
purpose, CD spectra were obtained after an overnight incubation of
approximately equimolar concentrations of syntaxin and full-length
SNAP-25. The CD spectrum of this complex was compared with the sum of
spectra (i.e. theoretical, noninteracting) that were
recorded for each individual protein, corrected for variations of the
protein concentrations (see "Experimental Procedures").
As shown in Fig. 5A, the CD spectrum of the
SNAP-25-syntaxin complex was clearly more
The observed increase in CD spectra of the complexes were also recorded in high salt (1 M NaCl; Fig. 5, D and E). When the
spectra were compared with the theoretical noninteracting sum of the
spectra of the individual components under these conditions, the
induced Although binding experiments have demonstrated that binding of
full-length SNAP-25 to syntaxin occurs with high affinity, it cannot be
excluded that dynamic equilibria exist which may contribute to the
spectrum. For these reasons, the binary complex was purified by ion
exchange chromatography, and its homogeneity was verified by size
exclusion chromatography. No measurable dissociation occurred during
purification (data not shown). The purified complex has a CD spectrum
that is very similar to that of the complex formed directly from its
constituents without further purification (compare Fig. 5, F
and D), although the molar ellipticities are somewhat
higher. This indicates that in the mixing experiments the resulting CD
spectrum is determined mainly by the complex.
No direct binding of a COOH-terminal fragment of SNAP-25 to syntaxin
has been described in previous studies (9, 13). This suggests that the
COOH-terminal fragment of SNAP-25 might bind only when a complex
between syntaxin and the NH2-terminal fragment of SNAP-25
has formed. To confirm this idea, GST-syntaxin, immobilized on
glutathione-Sepharose, was incubated sequentially with
SNAP-251-90wt and SNAP-25112-212. As shown in Fig. 6A, SNAP-25112-212 bound to
syntaxin when SNAP-251-90wt was present, whereas no
binding was observed to syntaxin alone. A similar result was obtained
when GST-SNAP-251-90wt was immobilized. Again, binding of
SNAP-25112-212 was dependent on the presence of syntaxin
(Fig. 6B).
In the
last series of experiments, amino acid substitutions were introduced in
the NH2-terminal fragment of SNAP-25 to investigate how
changes in the ability to bind syntaxin correlate with structural changes observed by CD. The first mutation consisted of a glycine to
aspartic acid substitution in the highly conserved (cf. Fig. 8) position 51 (SNAP-251-90GD). This substitution was
motivated by a similar exchange at the homologous position in the yeast Sec9-protein which results in a temperature-dependent loss
of function phenotype (25). Sec9 is an essential gene in
yeast which is required for the final exocytotic step in the
constitutive membrane trafficking pathway. Although Sec9p is
considerably larger than SNAP-25, the COOH-terminal domain, which is
homologous to SNAP-25, is apparently sufficient to sustain wild type
function (25), suggesting that Sec9 is a member of the SNAP-25 protein family. As shown in Fig. 7A, the
SNAP-251-90GD variant was unable to bind to syntaxin.
In the second set of mutations, either one or both of two conserved
hydrophobic amino acids, Met-40 and Met-43, were replaced with
glutamate residues (SNAP-251-90M40E,
SNAP-251-90M43E, and SNAP-251-90EE). No
binding of these mutants to GST-syntaxin was observed (Fig.
7A). In parallel, we substituted both methionines with
leucines (SNAP-251-90LL), i.e. a hydrophobic
but structurally different amino acid with a comparable side chain
volume. In fact, several SNAP-25 variants contain leucine at position
43, whereas Met-40 is conserved (see Fig. 8). As shown
in Fig. 7A, the resultant mutant protein bound to syntaxin
in a manner indistinguishable from wild type.
The CD spectra of the mutant proteins had a shape similar to that of
SNAP-251-90wt, but they differed in their Upon interaction with syntaxin the binding mutant
SNAP-251-90LL exhibited an increase in The binding of syntaxin and SNAP-25 is thought to be part of a
sequence of protein-protein interactions that leads from vesicle docking to membrane fusion. Although an increasing number of these interactions are known and the participating protein domains have been
identified, no biophysical and structural information regarding these
interactions has been available up to now. The CD data presented here
demonstrate that the formation of a binary complex between syntaxin and
SNAP-25 is associated with a dramatic increase in Is the interaction between the two proteins mediated by direct
interaction between adjacent We have reanalyzed the binding domains of all hitherto reported
isoforms of syntaxin and SNAP-25 using two different algorithms (20-22). The results generally confirm the high scores reported earlier. However, there is considerable variation between different species and between the two algorithms, particularly for the
NH2-terminal portion of SNAP-25. In this domain, two sets
of heptad repeats were identified (13, see Fig. 8). Although both
algorithms yield a probability of 1.0 for coiled coil formation of the
second repeat in all species variants, the scores are much more
divergent for the first repeat (Lupas (20, 21), using a window size of
27 residues/paircoil (22): rat 1.0/0.68, goldfish 0.99/0.51,
Torpedo 0.44/0, Drosophila 0.36/0, leech 0.02/0).
Helical wheel projection of the two putative repeats (Fig. 8,
B and C) shows that according to the predictions
Met-40 and Met-43 of the SNAP-25 molecule would be located in the
hydrophobic core of a coiled coil. Their replacement with charged side
chains would be expected to destabilize a coiled coil interaction,
whereas their replacement with appropriate hydrophobic side chains
should have little or no effect. These predictions agree with our
experimental findings.
Our data show that not only the NH2-terminal domain of
SNAP-25, but also the COOH-terminal domain participates in binding to
syntaxin, although, unlike the NH2-terminal domain, it
cannot bind on its own. Binding of the NH2-terminal domain
results in an increase in We thank Luke M. Rice and Isaiah T. Arkin for
stimulating discussions and assistance with CD spectroscopy and the
coiled coil prediction programs and Phyllis I. Hanson for helpful
comments on this manuscript. The
Department of Pharmacology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-helicity of the
individual proteins and to gain insight into structural changes
associated with complex formation. Syntaxin displayed approximately
43%
-helical content. In contrast, the
-helical content of
SNAP-25 was low under physiological conditions. Formation of the
SNAP-25-syntaxin complex was associated with a dramatic increase in
-helicity. Interaction of a 90-residue NH2-terminal
fragment of SNAP-25 comprising the minimal syntaxin binding domain lead
to a similar but less pronounced increase in
-helicity. Single amino
acid replacements in the putative hydrophobic core of this fragment
with hydrophilic amino acids abolished the induced structural change
and disrupted the interaction monitored by binding assays. Replacements
with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.
-,
- (brain-specific),
and
-SNAP), and the ATPase NSF
(N-ethylmaleimide-sensitive fusion protein). SNAPs and NSF
apparently operate on all relatives of the
synaptobrevin/syntaxin/SNAP-25 protein families, which are therefore
commonly referred to as SNAREs (SNAP receptors). ATP hydrolysis by NSF
leads to disassembly of the synaptobrevin-syntaxin-SNAP-25-complex (6),
an effect that is associated with a different state of syntaxin (7,
8).
-helical content of a protein. Furthermore, it can be used to assess
changes of secondary structure which occur upon modification of the
environment or upon complex formation. The SNAP-25 and syntaxin
variants from the leech (Hirudo medicinalis) were used in
this study. Both are highly homologous to their mammalian
counterparts.
Molecular Cloning of SNAP-25 and Syntaxin from Hirudo
medicinalis
-Zap library prepared from the nerve cord of the leech. The
leech variants of the synaptic proteins SNAP-25 and syntaxin exhibit
high homology to their mammalian counterparts. A detailed description
of the cloning strategy and the nucleotide sequences will be published elsewhere.2 For the bacterial expression of
recombinant proteins, full-length and truncated coding sequences were
amplified using the polymerase chain reaction with oligonucleotides
containing BamHI and EcoRI restriction sites and
subcloned into pGEX4-T (Pharmacia) or pTrcHisA (Invitrogen).
-CCGGGATCCATGGCCAAGGATATCAAG-3 and 5
-GCGGAATTCTTATTCTTTCAGGAGTTTGC-3
, respectively. For the generation of the 3
deletion mutant SNAP-251-90wt the primer
5
-GCGGAATTCTTATTCCATCCCTTCCAGGTT-3
was used. For the generation of
the 5
deletion mutant SNAP-25112-212 the primer
5
-GCGGGATCCTGGAACAAGGGCGACGAGGGA-3
was used. Leech syntaxin (residues
1-271, comprising the entire cytoplasmic domain) was constructed using
primers that were complementary to codons of amino acids 1-6 and
266-271. Site-directed mutagenesis of SNAP-251-90 (see
Fig. 8) was performed using the overlapping primer method (16). All
mutants were confirmed by resequencing the entire coding region.
Fig. 8.
The binding region of SNAP-25 to syntaxin
contains two putative coiled coil domains. Panel A, sequence
alignment of the 1-90 NH2-terminal fragment of SNAP-25.
Numbering refers to the leech sequence. Identities are on a
closed black background. Conservative exchanges of
hydrophobic and charged amino acids are on a gray shaded
background. The mouse sequence is shown as the exon 5b splicing
product, which is identical in all tetrapoda SNAP-25 (the GenBank
accession number is P13795[GenBank]). The goldfish (Carassius
auratus) sequence represents the isoform SNAP-25A (P36977[GenBank]). The
GenBank accession number for Torpedo marmorata is P36976[GenBank] and
for Drosophila melanogaster, P36975[GenBank]. Panels B and
C, the sequence of SNAP-251-90wt is projected onto two helical wheels as predicted by the Lupas (20, 21) and the
paircoil (22) algorithm. The first heptad repeat (panel B)
is predicted to start with Thr-17 in position f and to end with Cys-50 in position e. The second heptad repeat
(panel C) starts with Leu-58 in position a and
ends with Glu-90 in position e. The stretch between Gly-51
and Met-57 is not shown. The positions of the introduced point
mutations are boxed.
[View Larger Version of this Image (39K GIF file)]
]sum = (c1n1[
]1+
c2n2[
]2)/(c1n1 + c2n2), where
c1 and c2 are the
respective concentrations of peptide molarity, n1 and n2 are respective
number of amino acids, and [
]1 and
[
]2 are the observed mean residue ellipticities of the
two proteins. The CD spectrum of the purified SNAP-25-syntaxin complex
was recorded in 10 mM Tris, pH 7.4, 300 mM
NaCl, 1 mM EDTA, 1 mM DTT. The molar ellipticity was calculated assuming a 1:1 complex.
-helical content for each protein was calculated
using the assumption that for 100%
-helix the mean residue ellipticity, [
], at 222 nm is [
]222=
36,300
(1-2.57/X), where X is the number of amino acids
in the protein (19).
-mercaptoethanol) and heated for 5 min at 95 °C, subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis using T7-tagged monoclonal antibody (Novagen). The blots were stained with an alkaline phosphatase-conjugated secondary antibody using nitro blue tetrazolinum and
5-bromo-4-chloro-3-indolyl phosphate.
CD Spectroscopy of Syntaxin and SNAP-25
-helical content of approximately 43%. In
contrast, the CD spectrum of SNAP-25 indicates little
-helical
content (approximately 14%) and is reminiscent of partially
unstructured proteins (23).
Fig. 1.
CD spectra of syntaxin, SNAP-25, and SNAP-25
fragments at standard conditions. Panel A, CD spectra of
full-length SNAP-25 and syntaxin1-271. Panel B,
CD spectra of SNAP-251-90wt and
SNAP-25112-212. Panel C, mean residue
ellipticity ([]) at 222, 208, and 203 nm for different
concentrations of SNAP-251-90wt.
[View Larger Version of this Image (17K GIF file)]
-helical content (approximately 20%) than the full-length protein (Fig. 1B). The CD spectrum of SNAP-251-90wt did
not change when the protein concentration was varied more than 100-fold
(ranging from 0.3 to 36 mM, Fig. 1C). A fragment
of the COOH-terminal half of SNAP-25 (SNAP-25112-212)
exhibited a CD spectrum similar to that of the full-length protein,
with an
-helical content of approximately 13% (Fig.
1B).
-helicity of SNAP-251-90wt (Fig.
2, A and B), with minima at 208 and 222 nm clearly visible already at a concentration of 300 mM NaCl. The
-helical content in 1 M NaCl
was about 44%. To investigate if the increased
-helical content at
higher ionic strength was associated with a change in the oligomeric
state of the molecule, samples were analyzed by size exclusion
chromatography. In 100 mM NaCl, SNAP-251-90wt
eluted in two peaks with apparent molecular masses of 50 and 90 kDa. At
500 mM NaCl the molecule eluted in two peaks with apparent
molecular masses of 50 and 160 kDa. When the NaCl3
concentration was raised to 1 M, the protein eluted as a
single peak of molecular mass of 160 kDa (Fig. 2C).
Apparently, high ionic strength induced the formation of a defined
higher order oligomer with apparent molecular mass of 160 kDa, whereas
in low salt the protein structure is undefined, and the two peaks may correspond to different oligomeric species. The view that
SNAP-251-90wt is more structured in high salt was further
supported by limited tryptic digestion of the protein. In high salt,
only partial digestion was observed, resulting in a defined fragment of
reduced size, whereas in 100 mM NaCl the protein was
digested into small fragments (not shown).
Fig. 2.
Increase of -helicity and change of the
oligomeric state of SNAP-251-90wt by increasing
concentrations of NaCl. In the experiments standard conditions
were used except that NaCl concentrations were adjusted as indicated.
Panel A, effect of increasing NaCl concentrations on the CD
spectra of SNAP-251-90wt. Panel B, the mean
residue ellipticity ([
]) at 222 nm of SNAP-251-90wt spectra is plotted against NaCl concentrations. Panel C,
size exclusion chromatography of SNAP-251-90wt at
different NaCl concentrations. A Superdex-200 column was loaded with
200 µl of SNAP-251-90wt (approximately 10 µM) at 25 °C in 10 mM sodium phosphate
buffer at the NaCl concentrations indicated and eluted with the same
buffer. Column effluent was monitored at an OD of 280 nm.
Arrows indicate the positions of globular proteins used as
molecular mass standards (see "Experimental Procedures").
[View Larger Version of this Image (19K GIF file)]
-helical structure in SNAP-251-90wt (Fig. 3) with both Mg2+ and Ca2+
being effective. This potency is only partially attributable to the
increased ionic strength because similar concentrations of divalent
anions were ineffective (not shown). Rather, it may be due to more
efficient shielding of charges or formation of salt bridges between
negatively charged residues.
Fig. 3.
Increase of -helicity of
SNAP-251-90wt induced by increasing concentrations of
divalent cations. All recordings were done under standard
conditions supplemented as indicated. When CaCl2 was added
the phosphate buffer was replaced with 10 mM Tris-Cl, pH
7.4. Panel A, effect of MgCl2 concentration on the CD spectra of SNAP-251-90wt. Panel B,
effect of CaCl2 concentration on the CD spectra of
SNAP-251-90wt. Panel C, the mean residue
ellipticity ([
]) at 222 nm of SNAP-251-90wt spectra
is plotted against MgCl2 and CaCl2
concentrations.
[View Larger Version of this Image (23K GIF file)]
-helical content in SNAP-251-90wt. At pH
3 the
-helical content increased even further. This may be explained
by the fact that SNAP-251-90wt is negatively charged at
neutral pH (calculated pI = 4.55 including the His6
tag; note also that the protein was insoluble at pH values around its
isoelectric point). Negatively charged groups become protonated at low
pH, thus neutralizing potential charge-charge repulsions (24).
Fig. 4.
Changes in the CD spectra of
SNAP-251-90wt caused by variations of the pH.
Recordings were obtained in 100 mM sodium phosphate,
pH-adjusted with NaOH, containing 100 mM NaCl. Panel
A, effect of pH on the CD spectra of SNAP-251-90wt. Panel B, the mean residue ellipticity ([]) at 222 nm of
SNAP-251-90wt spectra is plotted against pH.
[View Larger Version of this Image (23K GIF file)]
-helical content of full-length SNAP-25 was
somewhat lower than that of the NH2-terminal fragment SNAP-251-90. The increase in its
-helical content in
high salt or at low pH was also less pronounced (19% in 1 M NaCl; 38% at pH 3) than for the NH2-terminal
fragment. When the COOH-terminal fragment of SNAP-25
(SNAP-25112-212) was analyzed at high ionic strength or at
low pH, no increase in
-helical content was observed (not shown).
This suggests that the
-helical structure induced by these
environmental changes is confined to the NH2-terminal half
of the molecule.
-helical than the
theoretical noninteracting sum of the individual spectra, demonstrating
that the complex had a higher
-helical content than the individual
components alone. A similar but less pronounced increase was observed
when the NH2-terminal fragment of SNAP-25
(SNAP-251-90wt) instead of the full-length protein (Fig.
5B) was used in the binding reaction. In contrast, no
increase in
-helicity was observed upon mixing of the COOH-terminal
half of SNAP-25 (SNAP-25112-212) with syntaxin (Fig.
5C).
Fig. 5.
Changes in the CD spectra caused by
interaction between SNAP-25 and syntaxin. If not indicated
otherwise, CD spectra were recorded in 10 mM sodium
phosphate, pH 7.4, 2 mM MgCl2, 1 mM
EGTA, 1 mM DTT, with the NaCl concentrations indicated in
the panels. Spectra of each individual binding partner were
recorded, and the sum of both spectra was calculated (dotted
line; corrected for variations of the protein concentrations; for
details, see "Experimental Procedures"). The spectra of the
combined components (solid line) were recorded after
overnight incubation of the binding partners. Panel A,
full-length SNAP-25 (8.7 µM) plus
syntaxin1-271 (7.1 µM) in 100 mM
NaCl. Panel B, SNAP-251-90wt (6.2 µM) plus syntaxin1-271 (7.1 µM) in 100 mM NaCl. Panel C,
SNAP-25112-212 (9.8 µM) plus
syntaxin1-271 (16.4 µM) in 100 mM NaCl. Panel D, full-length SNAP-25 (2.9 µM) plus syntaxin1-271 (2.7 µM) in 1 M NaCl. Panel E,
SNAP-251-90wt (3.1 µM) plus
syntaxin1-271 (2.7 µM) in 1 M
NaCl. Panel F, CD spectrum of the purified SNAP-25-syntaxin complex. Purification consisted of anion exchange chromatography. The
spectrum was recorded in 300 mM NaCl, 10 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM DTT at
25 °C.
[View Larger Version of this Image (35K GIF file)]
-helicity during complex formation may be
due to a change in SNAP-25 alone, in syntaxin alone, or in both
proteins. However, the CD spectrum of syntaxin did not change under any
of the environmental conditions tested (high salt, divalent ions, high
or low pH; not shown), whereas
-helicity could readily be induced in
SNAP-25. This suggests that the increase in
-helicity upon complex
formation is due to a change in SNAP-25.
-helicity was less pronounced in the SNAP-25-syntaxin
complex, and no induction was observed in the
SNAP-251-90wt-syntaxin complex. Since the induced
-helicity was always larger for the full-length SNAP-25-syntaxin complex than for the SNAP-251-90wt-syntaxin complex, it is
likely that the COOH-terminal half of SNAP-25 also becomes more
structured upon complex formation.
Fig. 6.
Binding of the COOH-terminal half of SNAP-25
to a complex of SNAP-251-90wt and syntaxin. Panel
A, 10 µg of GST-syntaxin1-271 immobilized on
glutathione-Sepharose was incubated overnight at 4 °C with 500 µl
of the indicated His6-tagged fragments of SNAP-25 (each
about 10 µM). Panel B, 10 µg of
GST-SNAP-251-90wt immobilized on glutathione-Sepharose was
incubated overnight at 4 °C with 500 µl the
His6-tagged proteins as indicated (each about 10 µM). Proteins bound to the beads were analyzed by an
immunoblot assay using the monoclonal T7-tagged antibody against a
recognition domain in the His6-tag (TrcHisA). The
NH2-terminal half of SNAP-25 binds to syntaxin, whereas the
COOH-terminal half of SNAP-25 only binds to a complex of the
NH2-terminal half of SNAP-25 and syntaxin.
[View Larger Version of this Image (22K GIF file)]
Fig. 7.
Mutants of SNAP-251-90: CD
spectroscopy and binding properties. Panel A, binding of
SNAP-251-90 fragments to
GST-syntaxin1-271 or GST, immobilized on
glutathione beads. The figure shows protein retained on the beads,
analyzed by immunoblotting (see Fig. 7 for details).
SNAP-251-90wt and SNAP-251-90LL bind
specifically to GST-syntaxin1-271, whereas the mutants
SNAP-251-90GD, SNAP-251-90EE, SNAP-251-90M40E, and SNAP-251-90M43E do not
bind to syntaxin. The mutants SNAP-251-90M40E and
SNAP-251-90M43E exhibit only nonspecific binding to
syntaxin. Panel B, CD spectra of mutated
SNAP-251-90 fragments in standard conditions. Panels
C-E, CD spectra were recorded in 10 mM sodium
phosphate, pH 7.4, 2 mM MgCl2, 1 mM
EGTA, 1 mM DTT. Panel C,
SNAP-251-90LL (6.8 µM) plus
syntaxin1-271 (7.1 µM). Panel D,
SNAP-251-90GD (8.1 µM) plus
syntaxin1-271 (7.1 µM). Panel E,
SNAP-251-90EE (5.8 µM) plus
syntaxin1-271 (7.1 µM).
[View Larger Version of this Image (42K GIF file)]
-helical content (Fig. 7B). SNAP-251-90LL showed higher
-helicity than wild type, whereas SNAP-251-90EE had
lower
-helicity (Fig. 7B), an effect that was less
pronounced for the single site mutants SNAP-251-90M40E and
SNAP-251-90M43E (not shown). Similarly, substitution of
Gly-51 with aspartic acid
(SNAP-251-90GD) reduced the
-helical
content (Fig. 7B). These differences between the
-helical
contents of the binding and nonbinding mutants became more pronounced
at high ionic strength or in the presence of divalent cations (not
shown). All SNAP-251-90 variants had a high
-helical
content at pH 3. Apparently, protonation of the glutamate (SNAP-251-90EE) or the aspartate side chains
(SNAP-251-90GD) restores the ability of these mutants to
form
-helices.
-helicity which
was very similar to that of the wild type fragment (Fig.
7C). In contrast, no difference between the measured and
calculated CD spectra was observed when the nonbinding mutants
(SNAP-251-90EE and SNAP-251-90GD) were mixed
with syntaxin (Fig. 7, D and E).
-helical content
which presumably occurs mainly in SNAP-25.
-helices? Several investigators noted
earlier that the minimal binding domains of both SNAP-25 and syntaxin
have a high propensity for the formation of coiled coils (9, 13, 14,
25) and suggested that binding is mediated by such interactions. The
dramatic increase in
-helical content upon complex formation is
consistent with an induced coiled coil formation, although alternative
structures cannot be excluded at present. Two-stranded coiled coils
consist of two amphipathic right-handed
-helices that are twisted
around each other, forming a left-handed superhelix (26). The
individual helices bury hydrophobic residues along one side of the
helix. These side chains form the hydrophobic core of the superhelix
and are often flanked by charged side chains that interact
electrostatically (24). Coiled coils frequently form the basis of
regulated protein-protein interactions, e.g. binding and
activation of bZIP transcription factors (27, 28).
-helicity which is similar to but less
pronounced than binding of the full-length protein. This difference
reflects a true structural difference and not merely a higher relative complex concentration since in 1 M NaCl an increase in
-helicity is only observed when full-length SNAP-25 is used. It is
likely that the COOH-terminal domain also undergoes a structural change upon binding, although other explanations cannot be ruled out. Previous
work has established that the COOH terminus of SNAP-25 is required for
binding of synaptobrevin to SNAP-25 (13) and, furthermore, that
formation of the SNAP-25-syntaxin complex greatly increases the
affinity for synaptobrevin beyond that of either partner alone (9, 11).
It is tempting to speculate that formation of the ternary complex is
mediated by a global structural change in SNAP-25 which in turn
provides an optimized attachment site for synaptobrevin.
*
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (to D. F.) and by National Institutes of Health Grant GM54160-01 (to A. T. B.). 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.
To whom correspondence should be sent: Dept. Molecular
Biophysics and Biochemistry, Bass Center, 266 Whitney Ave., New Haven, CT 06520. Tel.: 203-432-6143; Fax: 203-432-6946; E-mail:
brunger{at}laplace.csb.yale.edu.
1
The abbreviations used are: SNAP(s), soluble NSF
attachment protein(s); NSF, N-ethylmaleimide-sensitive
fusion protein; SNAP-25, synaptosomal associated protein of 25 kDa;
SNARE, SNAP receptor; DTT, dithiothreitol; GST, glutathione
S-transferase.
2
Bruns, D., Engers, S., Yang, C., Ossig, R.,
Jeromin, A., and Jahn, R. (1997) J. Neurosci., in
press.
3
Programs are available on the World Wide Web
(URLs: http://ulrec3.unil.ch/software/COILS_form.html and
http://ostrich.lcs.mit. edu/cgi-bin/score).
-Zap library prepared from nerve
cord of H. medicinalis was kindly provided by Roberta Allen
and Steve Heineman of the Salk Institute.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.