(Received for publication, October 25, 1996, and in revised form, December 3, 1996)
From the Departments of Chemistry, Synaptotagmin (Syt) II, a synaptic vesicle
protein containing two copies of highly conserved protein kinase C
homology regions known as the C2A and C2B domains, acts as a
Ca2+ sensor and provides both phospholipid and inositol
polyphosphate (IPn) recognition domains important in endo- and
exocytosis. Four photoaffinity analogues of IP3,
IP4, and IP6 containing a P-1- or P-2-linked
4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label
glutathione S-transferase (GST) fusion constructs of the
Syt II-C2A and C2B domains. The P-2-linked
[3H]BZDC-IP6 showed efficient,
IP6-displaceable labeling of the GST-Syt II-C2B. The rank
order of photocovalent modification paralleled the order of competitive
displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6
failed to label the C2B domains. The GST-Syt III-C2B domain, which
lacks IP6 binding affinity, also failed to undergo labeling
by P-2-linked [3H]BZDC-IP6. When mixtures of
the 32-amino acid basic peptide corresponding to the essential
IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B
were labeled by a stoichiometric amount of P-2-linked
[3H]BZDC-IP6, the two polypeptides showed
equivalent affinity for the photolabel. Although the CD spectrum of
this 32-mer at two pH values showed a random coil, the photoaffinity
analogue of IP6 appeared to induce a binding-compatible
structure in the short peptide.
The synaptotagmins (Syts)1 are
synaptic vesicle proteins that play essential roles in nucleating the
clathrin coat during endocytosis and in acting as Ca2+
sensors (1-3) and phosphoinositide sensors (4) during exocytosis. They
are a critical part of a complex machinery of intracellular protein
transport (5, 6) and the synaptic vesicle cycle (7). In addition to a
short N-terminal intravesicular region and single transmembrane domain,
Syts have two copies of highly conserved repeats, known as the C2A and
C2B domains, which are homologous to the C2 regulatory region of
protein kinase C (8). In particular, the C2B domain appears to play
several roles. First, the C2B domain of mouse Syt II shows specific
binding to high polyphosphate inositols (IPns) (9), a feature
that is not shared by the highly homologous C2A domain nor with the C2
domains of other proteins such as rabphilin. Moreover, the C2B domain
is necessary but not sufficient for IPn binding. Although Syt
II and IV show high affinity binding of IP4 and
IP6, the Syt III-C2B domain shows negligible binding,
despite a high sequence identity with the Syt II-C2B domain, including
the 32-residue region examined by mutational analysis (10). Inhibition
of C2B function in the squid giant axon disrupts synaptic vesicle
release and recycling (11). Similarly, Caenorhabditis
elegans mutants lacking Syt or simply the C2B domain cannot
recycle synaptic vesicles (12), and the presence of Syt I in
cerebrospinal fluid of Alzheimer's patients (13) may provide clues to
the disruption of synaptic function in humans. The C2B domain acts as a
high affinity receptor for clathrin assembly protein AP-2 (14), and
Ca2+ appears to mediate Syt dimerization via the C2B domain
(15).
Synaptotagmin II isolated from mouse cerebellum shows high affinity
binding to Ins(1,3,4,5)P4 with a KD of
30 µM (16) and 117 nM for the GST-Syt II-C2B
construct (9). Curiously, the rank order of IPns binding for
native Syt II was Ins(1,3,4,5,6)-P5 > (1,3,4,5)-P4 > Ins(1,2,3,4,5,6)-P6 > Ins(1,4,5)-P3, whereas for the GST-Syt II-C2B domain the
order was IP6 > IP5 > IP4 > IP3. Deletion mutants allowed mapping of the IPn binding site to the central region of the mouse Syt II-C2B domain, specifically residues 315-346 (IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF) (9). In order to obtain direct evidence for the binding site of
IPns in the C2B domain, to examine the InsPn selectivity of the domains, to explore the Ca2+ dependence
of binding, to determine the role of the central 315-346 peptide, and
to evaluate the reason for the failure of Syt III-C2B domain to bind
IPns, we undertook a series of photoaffinity labeling
experiments using four tritium-labeled, benzophenone-containing derivatives of IP3, IP4, and IP6
(17).
P-1-Tethered
[3H]BZDC-IP3 was prepared by coupling the
1-O-(3-aminopropyl)-D-myo-Ins(1,4,5)P3
(18, 19) with [3H]BZDC-NHS ester (20). P-1-tethered
[3H]BZDC-IP4 (21) was prepared analogously
from
1-O-(3-aminopropyl)-D-myo-Ins(1,3,4,5)P4 (22). P-1-O-(6-aminopropyl)-IP6 and its
[3H]BZDC photoaffinity label were prepared as described
elsewhere (23). Specific activities for all BZDC derivatives were
35-42 Ci/mmol, depending on the lot of [3H]BZDC-NHS
ester used. D-myo-IP3 and
IP4 were synthesized (18, 22), and IP6 was
obtained from Sigma. All other chemicals were commercial products of reagent grade. Solutions were made in nanopure (ultrafiltered, distilled, and deionized) water. The peptide
IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF was synthesized by the Peptide
Institute (Osaka, Japan) and was >98.1% homogeneous by HPLC, YMCPak
ODS-AM, 10-60% CH3CN in 0.1% trifluoroacetic acid.
2-O-(6-Aminohexyl)-meso-Ins(1,2,3,4,5,6)P6
was prepared as described (24) and coupled with
[3H]BZDC-NHS in aqueous
N,N-dimethylformamide (20). The following protocol was typical for several independent preparations of this photoactivatable ligand. [3H]BZDC-NHS (2 mCi, 0.05 µmol) was dissolved in N,N-dimethylformamide (50 µl) and a solution of 1 molar equivalent of
aminohexyl-IP6 (50 µg, 0.05 µmol) in 0.25 M
triethylammonium bicarbonate buffer (50 µl, pH 8.0) was added. The
reaction was stirred overnight, concentrated in vacuo,
redissolved in water, and loaded onto a DEAE-cellulose column
(HCO3 JM109
Escherichia coli cells were electroporated and transformed
with purified plasmids as described (9). Cells were grown in
LB-ampicillin and induced with
isopropyl-1-thio- GST fusion
proteins (1 µM) were incubated with 30 µl of
phosphate-buffered saline (pH 7.4) containing 0.5 µCi (0.25 µM) of [3H]BZDC-IPn
(n = 3, 4, or 6). To determine the affinity and
specificity of binding, unlabeled IPn (0.25 mM) was included in the solution. Samples were equilibrated at 4 °C for 15 min in a 96-well plate. Wells in the plate were aligned with the axis
of a UV light with a 2-cm distance between the bottom of the wells and
the bulb and irradiated (360 nm at 1900 µW/cm2) for 45 min at 0 °C. Then 6 × SDS sample buffer was added to the
samples, and the proteins were separated by SDS-polyacrylamide gel
electrophoresis on 12.5% Laemmli gels. The gels were stained with
Coomassie Blue, destained, and impregnated with En3Hance
(DuPont NEN) according to the manufacturer's instructions, dried, and
exposed to XAR-5 x-ray film for 5-30 days at CD spectra were recorded using an AVIV
model 62DS spectropolarimeter. Far-UV CD spectra of 50 µM
peptide solutions were recorded at 11 °C using a 1-mm quartz
cell.
Four photoaffinity probes
(Fig. 1) were used to probe the selectivity and
specificity of the GST-Syt II-C2B interaction with IPns. Each
inositol polyphosphate photoaffinity label (25) was freshly prepared
for this study from the corresponding 1- or
2-O-(
To determine the
selectivity of the BZDC-IPn probes for Syt II domains, the
crude supernatant proteins from transformed E. coli were
photolabeled with [3H]BZDC-IP4 and
[3H]BZDC-IP6. To ascertain whether any
nonspecifically or specifically labeled protein was derived from
plasmid or from E. coli, we also photolabeled a crude
supernatant containing a GST fusion to a truncated human thrombin
receptor.2 Fig. 2 shows the
initial labeling experiments. Neither the 40-kDa GST-C2B nor the 57-kDa
GST-C2A+C2B protein bands (arrows) were labeled by
[3H]BZDC-IP4 in the crude supernatants.
Interestingly, an ~45-kDa protein present in all supernatants,
including the GST-NTTR control, showed efficient and specific
IP4-displaceable labeling. This observation suggests the
presence of a hitherto unknown bacterial IP4-binding
protein. In contrast, [3H]BZDC-IP6
efficiently and selectively labeled both the 40-kDa GST-Syt II-C2B and
57-kDa GST-Syt II-C2A+C2B proteins, with no detectable labeling of
proteins in the GST-NTTR control. It is noteworthy that despite the low
abundance of the GST-Syt II-C2B constructs in the crude supernatants,
virtually no other proteins were labeled. The specificity of the
labeling was confirmed by the absence of labeling in the presence of
1000-fold excess IP6 as a competitor. Initial data
supported a higher affinity of the Syt II-C2B constructs for
[3H]BZDC-IP6 relative to
[3H]BZDC-IP4 consistent with binding affinity
measurements (9).
To study
the relative specificity for different IPns, the GST-Syt II-C2B
fusion protein was purified by glutathione affinity chromatography and
incubated with 0.25 µM
[3H]BZDC-IP6 in the presence of increasing
concentrations of IP3, IP4, and
IP6. The unlabeled IPns should compete for the same
binding pocket with the labeled BZDC-IP6. After a 10-min preincubation, irradiation (45 min, 4 °C, 360 nm), followed by electrophoresis and fluorography, provided the result shown in Fig.
3A. The data show substantial qualitative
differences. At 1000-fold excess, IP3 fails to displace the
labeling, IP4 shows ~60% reduction of labeling, and
IP6 completely displaces labeling by
[3H]BZDC-IP6; over 90% reduction in labeling
is seen with 250-fold excess IP6.
The fusion protein GST-Syt II-C2B was
irradiated for 20, 30, 45, 60, and 90 min at 4 °C with
[3H]BZDC-IP6 to determine the optimal time
for covalent attachment of the ligand to the protein. As expected from
previous studies with benzophenone (26) and specifically
[3H]BZDC-modified-(P)IPns (17), the covalent
binding reached saturation at 45 min. After 45 min, the specific
binding sites were saturated and only nonspecific labeling occurred.
This validated the selection of 45 min for the screening studies
described above; in addition, all subsequent photolabeling experiments
employed 45-min irradiation times.
To determine the specificity of the Syt II-C2B domain for different
IPns, the purified GST-Syt II-C2B fusion protein was
photoaffinity labeled with each of the three
[3H]BZDC-IPn probes (Fig. 3B).
Consistent with data from the photolabeling of crude supernatants from
transformed cells, the purified fusion protein showed maximal binding
for P-2-tethered [3H]BZDC-IP6 with somewhat
reduced labeling by [3H]BZDC-IP4. Labeling by
[3H]BZDC-IP3 incorporation is over four times
lower when compared with P-2-linked
[3H]BZDC-IP6. Unexpectedly, no detectable
labeling was observed with the P-1-linked [3H]BZDC-IP6.
These data suggest three key interpretations: 1) the presence of a
phosphate group at the 3 position of the inositol group substantially
increases the binding; 2) the presence of phosphates at all 6 positions
may lead to better binding when compared with IP4, and 3)
both the position of tethering and number of phosphates are important
in determining binding affinity and covalent attachment.
Based on
deletion and truncation constructs of the Syt II-C2B domain, a
lysine-rich region
(315IHLMQNGKRLKK To determine the relative affinities of the complete C2B domain and the
32-mer for IPn binding, the purified GST-Syt II-C2B domain was
photoaffinity labeled with [3H]BZDC-IP6 in
the presence of 0, 1, 2, 4, 8, and 16 molar equivalents of this peptide
(Fig. 4). At equimolar concentrations of GST-Syt II-C2B
and the peptide, labeling of the complete domain and the central 32-mer
are essentially equal. Labeling of the C2B fusion protein decreases and
the labeling of the 32-mer increases as the peptide concentration is
increased. At 4 equivalents of the peptide, nearly 95% of the
[3H]BZDC-IP6 is sequestered by the 32-mer,
thereby "protecting" the C2B fusion protein from labeling. At 8 and
16 equivalents of peptide (>20 µM), aggregation was
observed during electrophoresis (data not shown).
Because the peptide showed an affinity for P-2-tethered
[3H]BZDC-IP6 nearly equal to that of the
intact C2B domain, CD spectra were obtained to determine whether stable
secondary structures could account for these observations. Thus, CD
spectra were recorded on 50 µM solutions in aqueous
buffers (145 mM phosphate-buffered saline, pH 7.4, and in
0.5 mM acetic acid, pH 4.6) and were consistent with a
random coil, suggesting that the peptide had no long-lived secondary
structure under these conditions. As illustrated in Fig.
5, the addition of 0.1, 0.5, or 1 equivalent of
IP6 to the pH 7.4 solution failed to alter the random coil
nature of the CD spectrum.
Synaptotagmins have two C2
domains, C2A and C2B, but only the C2B domain binds to IPns.
The C2A domain appears to acts a Ca2+ sensor (1, 3) and
binds to phospholipids in a Ca2+-dependent
manner (27). To confirm the selectivity of
[3H]BZDC-IP6 for the C2B domain, three GST
fusion constructs (9) containing the C2B, the C2A, and the C2A+C2B
domains of Syt II (Fig. 6A) were expressed,
purified, and photoaffinity labeled. Consistent with the known
specificity of the C2B, but not C2A, domain for high affinity binding
of [3H]IP4 and higher IPns (9), only
the C2B and C2A+C2B domains were covalently modified with
[3H]BZDC-IP6. No photoaffinity labeling of
the C2A domain was detected.
Different isoforms of synaptotagmin have been found in mammals (2, 28,
29). Curiously, despite significant sequence similarity in the C2B
domains and particularly in the central base-rich IPn binding
region (Fig. 6B), the Syt III-C2B domain did not show high
affinity binding of [3H]IP4 (10). This
appeared to involve masking of the active site by the C-terminal
region, because truncation of the C-terminal one-third of Syt III-C2B
domain resulted in recovery of 30% of the
[3H]IP4 binding. Moreover, a chimeric fusion
protein, GST-Syt III/II-C2B, containing the C-terminal third of Syt
II-C2B domain fused to the N-terminal two-thirds of the Syt III-C2B
domain also recovered 70% of the [3H]IP4
binding (10). Thus, these two GST fusion proteins (see Fig.
6A) were expressed, purified, and photoaffinity labeled with [3H]BZDC-IP6 (Fig. 6C). Consistent
with its failure to bind [3H]IP4 under
equilibrium conditions, GST-Syt III-C2B domain showed negligible
photoaffinity labeling by [3H]BZDC-IP6. The
truncated GST-Syt III-C2B( The availability of tetherable analogues of the inositol
polyphosphates and phosphoinositide polyphosphates (25), including affinity purification resins and benzophenone-containing photoaffinity labels (17, 26) has enabled the identification of new PIPn- and
IPn-binding proteins and the characterization of the ligand
binding sites within these proteins. For example, four IP4
binding proteins were affinity-purified from rat brain (30) and
photoaffinity labeled with [3H]BZDC-IP4 (22,
31). Two have been further characterized; the Six GST-Syt C2 domain-containing fusion proteins were examined in this
study, using four photoaffinity probes corresponding to modifications
of IP3, IP4, and IP6. The most
important findings are summarized as follows. First, we observed
extremely high selectivity and ligand specificity of labeling of the
C2B-containing proteins in crude extracts. No other proteins were
significantly labeled with the high specific activity
[3H]BZDC-IPn derivatives even when the target
proteins represented less than 2% of the total protein. The only
exception was a hitherto unknown 45-kDa E. coli protein that
showed high selectivity and specificity for binding of IP4.
This observation may provide the basis for purification of this protein
and the determination of its role in the biochemistry of this common
bacterium. For all proteins and ligands, labeling followed an expected
time course, with a maximum efficiency at 45 min.
Second, labeling was highly dependent on the choice of ligand. Although
P-2-linked [3H]BZDC-IP6 gave the strongest
labeling of C2B-containing constructs, P-1-linked
[3H]BZDC-IP4,
[3H]BZDC-IP3, and
[3H]BZDC-IP6 showed modest, weak, and no
labeling, respectively. The failure of the P-1-tethered IP6
to label C2B domains was unexpected but an important "negative
control." It is important to recognize that the
aminopropyl-tethered photophore adds significant "unnatural" lipophilicity to the soluble IPn recognition elements as well
as masking one of the phosphates. The importance of the number and the
positions of phosphates and the position of the linker were critical
factors determining the photolabeling efficiency, not simply
nonselective hydrophobic interactions of the photophore with the
protein.
Nonetheless, the observation that purified but not crude C2B domains
could be weakly labeled by [3H]BZDC-IP3
emphasizes two important points. When only a single protein is present
in a photoaffinity labeling experiment, even relatively low affinity
sites may become covalently modified. The ligand may poorly associate
with the target, but in the absence of alternative sites for occupancy,
the irreversible nature of the photoaffinity labeling experiment allows
labeled protein to accumulate.
The photoaffinity probe may also mimic a different ligand. In
BZDC-IP3, the presence of the
P-1-O-(3-aminopropyl) linker bearing the hydrophobic
tritiated 4-benzoyldihydrocinnamide photophore allows the photoaffinity
ligand to resemble a 2-desacyl phosphoinositide 4,5-bisphosphate,
PIP2. For example, profilin, a PIP2-binding cytosolic protein important in interactions of the actin cytoskeleton with membrane phospholipids (36) has been labeled with
[3H]BZDC-IP3, but displacement of labeling
requires PIP2, not IP3 (17). The PH domain of
the phospholipase C Similarly the [3H]BZDC-IP4 probe effectively
mimics PIP3. This observation in fact enabled the isolation
and purification of centaurin- The relative importance of the phosphate groups in binding to the C2B
domains was examined in an independent set of NMR studies (40). The
relative rate enhancements, which depend on increases in motional
correlation time, were determined for clearly resolved phosphorus and
proton signals of the photoaffinity ligand in the presence and the
absence of GST-Syt II-C2B. Thus, 1H and 31P NMR
of 10 mM BZDC-IP4 were first determined at 298 K for 20 mM sodium acetate buffer containing 0.1 mM EDTA, pH 5.5; next, 0.04 molar equivalent of purified
GST-Syt II-C2B (0.4 mM) was added to the solution, and the
chemical shifts and relaxation times were remeasured. The signals
exhibiting the largest rate enhancements included P-5, H-5, and H-6,
whereas the P-1 and side chain aminopropyl relaxation rates were
essentially unaffected by the binding to the C2B domain. Thus, the
4,5,6 region of the inositol ring is intimately involved in the binding
to C2B, whereas the aminopropyl tether retains motional freedom
even in the bound state (40).
Third, labeling was well correlated with reported relative IPn
binding affinity as determined by equilibrium methods using
displacement of [3H]IP4 with unlabeled
IPns (9). Thus, using [3H]BZDC-IP6 as
the probe, displacement of 50% of the labeling required about a
150-fold molar excess of IP6, whereas little or no
displacement was observed with IP4 or IP3 at
six times this concentration of displacer. When
[3H]BZDC-IP4 labeling was examined in detail
(data not shown), IP6 was again the most effective
competing ligand.
Fourth, labeling was correlated with binding affinities of C2B domains
for the functionally diverse Syt II and Syt III constructs. Consistent
with the [3H]IP4 binding affinities (10), the
GST-Syt III-C2B domain was not covalently labeled upon irradiation with
[3H]BZDC-IP6. Moreover, purified GST fusion
proteins in which [3H]IP4 binding had been
partially restored also recovered the ability to be photoaffinity
labeled. Thus, the C-terminal truncated GST-Syt III-C2B( Fifth, labeling by P-2-linked [3H]BZDC-IP6
correlated with the known affinities of the two different C2 domains,
which show different calcium ion-dependent properties (41).
The C2A domain acts as a calcium sensor (3) during endocytosis, and
three-dimensional structures have been described (42, 43) for this
critical domain. The phospholipid selectivities of several Syts have
been described in detail, and the Syt IV C2A domain also appears to function as a calcium sensor (27). Consistent with the absence of high
affinity [3H]IP4 binding to C2A (10), the
GST-Syt II-C2A domain was not labeled by
[3H]BZDC-IP6. Preliminary observations
suggest Ca2+-dependent labeling of this domain
with [3H]BZDC-IP3, consistent with binding to
other anionic phospholipids such as phosphatidylserine. In contrast,
the C2B domain has been implicated in binding to IPns (9),
PIPns (38), AP-2 (14), and other elements of the synaptic
vesicle fusion machinery (44). The efficient labeling of the GST Syt II-C2B domain (and GST-Syt II-C2A+C2B domain) with
[3H]BZDC-IP6 is consistent with the different
affinities of these two protein kinase C2 homology domains.
Sixth, a synthetic 32-residue peptide corresponding to the base-rich
central region of the C2B domain, identified by mutational and
truncation/deletion methods as crucial to high affinity
[3H]IP4 binding (9, 10), also showed high
affinity for the [3H]BZDC-IP6 photoaffinity
probe. Indeed, when an equimolar ratio of 32-mer to GST-Syt II-C2B was
labeled with a stoichiometric amount of this photoaffinity label, the
full-length C2B domain and 32-mer were equally labeled. Increasing
amounts of peptide were able to completely sequester the photoprobe
from the C2B domain. This observation is highly significant in that it
represents the smallest minidomain known to demonstrate selective, high
affinity binding to IP6 or its analogues. Surprisingly,
this peptide failed to exhibit secondary structure, other than random
coil, in aqueous buffers at two pH values in the presence and the
absence of IP6. It is conceivable, in view of the
dimerization of Syts via their C2B domains (15), that the peptide is
only able to adopt an IP6-binding conformation in the
presence of a preorganized IP6-C2B complex. However, the
32-mer peptide could be labeled successfully with
[3H]BZDC-IP6 even in the absence of the GST
Syt II-C2B protein (data not shown), and this labeling can be reduced
by addition of IP6 as a competitor. This suggests that
BZDC-IP6 may induce a structure in the otherwise random
coil polypeptide. The labeling was maximal with
[3H]BZDC-IP6 relative to the
[3H]BZDC-IP3 and IP4 photoprobes,
supporting the higher affinity of the 32-mer peptide for
IP6.
A related C2B domain has been identified in a novel
IP4-binding protein from rat brain and both human and
porcine platelets (45); this protein also possesses GTPase-activating
activity not found in other IP4-binding proteins. This
protein has high specificity for IP4, with 1000-fold lower
affinity for IP6. The central IPn binding sequence
would have missing or moved basic residues found in the IP6
binding region of C2B with a central region of EAKKTKVKKK (human) as
opposed to KKKKTTVKKK (mouse). A preliminary report suggested that a
25-mer peptide that includes this recognition site could bind
IP4 with high affinity via a "basket-like" structure,
allowing the lysines or arginines to interact with the phosphates of
the IP4 (46). The data described herein constitute the
experimental evidence in support of this hypothesis.
Synaptotagmin is essential for several aspects of synaptic vesicle
dynamics. In particular, strong biological evidence now supports a
critical role for the C2B domain in recycling of synaptic vesicles.
This evidence is based on the accumulation of synaptic vesicles in
C. elegans mutants lacking the C2B domain of their Syt (12)
and from the inhibition of synaptic vesicle recycling in squid
(Loglio pealei) giant synapse preterminals when injected with antibodies to the this C2B domain (11). The high inositol polyphosphates (IPns, n = 4, 5, or 6) can block
synaptic neurotransmitter release and may regulate the fusion step
preceding exocytosis (11). The emergence of PIP3 as a
preferred ligand for inhibition of clathrin coating mediated by AP-2
(38) and AP-35 may suggest that the IPns
(n = 4, 5, or 6) may not be the actual physiological
ligands involved. An important research area would now be studies
directed at determining the nature and flux of PIPn and
IPn ligands actually involved in the endo- and exocytotic
events in vivo.
Four photoaffinity analogues of IP3,
IP4, and IP6 have been employed to document the
selectivity and specificity of different inositol polyphosphates toward
the C2 domains of two Syts. We made five key observations. First,
labeling of the C2B-containing proteins in crude extracts showed
extremely high selectivity and ligand specificity. Second, labeling was
highly dependent on ligand, with [3H]BZDC-IP6
showing the optimal labeling of C2B domains. Third, labeling was well
correlated with reported relative IPn binding affinities as
determined by equilibrium methods using displacement of
[3H]IP4 with unlabeled IPns. This
validates photolabeling as a methodology for identification of novel
proteins that bind IPns. Fourth, labeling was correlated with
[3H]IP4 binding affinities of C2B domains for
the functionally diverse Syt II and Syt III constructs and for the C2A
versus C2B domains of Syt II. Fifth, a 32-residue base-rich
peptide located in the central region of the C2B domain exhibited high
affinity binding to the photoaffinity label when employed in
competition with the full-length C2B domain. This observation opens the
door to use of peptides in physiological experiments to modulate
IPn-receptor binding in a wide variety of systems. Future
studies on active site mapping and structure determination of
domain-IPn complexes will also be realizable goals given these
new data.
We thank Dr. G. Dormán (Stony Brook,
NY) for advice. The [3H]BZDC-NHS ester reagent was
generously provided by Dr. D. G. Ahern, NEN Life Sciences (Boston,
MA).
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
Chemicals
form). The column was eluted
with a step gradient of 0.1 to 1.0 M triethylammonium
bicarbonate buffer; the [3H]BZDC-IPn was eluted
at 0.4-0.5 M triethylammonium bicarbonate buffer. Its
purity was analyzed by reverse phase HPLC (Aquapore RP-300, 7 µm,
4.5 × 250 mm, Brownlee column) using UV (254 nm) and
radiochemical detection. The pure [3H]BZDC-IPn
eluted at a retention time of 13 min using 0.05 N potassium
phosphate in 15% acetonitrile water.
-D-galactopyranoside, and expressed
proteins were isolated as described (9, 10). GST fusion proteins were
purified by chromatography on glutathione-Sepharose 4B (Pharmacia
Biotech Inc.) as specified by the supplier.
80 °C. No covalent
incorporation of the photolabel occurred in the absence of UV
irradiation. All of the photolabeling experiments were performed in at
least three independent replicates. Fluorograms were digitized on a
UMAX-UC 840 scanner using Adobe Photoshop, and the densities of the
bands were determined by NIH IMAGE 1.59 to find the relative incorporation.
Synthesis of Photoaffinity Labels
-aminoalkyl) IPn derivative with the
heterobifunctional reagent [3H]BZDC-NHS ester (20). The
P-1-linked 3-aminopropylphosphodiesters of
D-myo-IP3 (18) and IP4
(22), and the P-2-linked 6-aminohexylphosphodiester of
meso-IP6 (24) were prepared as described
previously. The racemic P-1-linked 6-aminohexyl derivative of
IP6 was prepared by modification of the protecting
group strategy developed for the P-2-linked probe (23). Each probe had
the same nominal specific activity, between 35 and 42 Ci/mmol, as
determined by the specific activity of the lot of
[3H]BZDC-NHS ester employed. This ensured that a given
level of radioactivity (in dpm) corresponded to an equivalent
concentration of each probe, so that direct comparisons of
relative efficiency of photoattachment would be meaningful. The
homogeneity of each probe was assessed by reverse phase HPLC following
periodic repurification of [3H]BZDC-IPns by ion
exchange chromatography.
Fig. 1.
Structures of inositol polyphosphate
photoaffinity probes.
[View Larger Version of this Image (19K GIF file)]
Fig. 2.
Photoaffinity labeling of crude supernatant
proteins from E. coli cells transformed with GST-Syt
II-C2B, GST-Syt II-C2A+C2B and a control plasmid encoding a GST fusion
with the soluble N-terminal domain of the human thrombin receptor
(NTTR). A, SDS-10% polyacrylamide gel
electrophoresis gel stained with Coomassie Blue. The arrows indicate positions of recombinant fusion proteins. B,
corresponding fluorogram of
[3H]BZDC-IP4-labeled proteins. C,
fluorogram of [3H]BZDC-IP6-labeled proteins.
The crude supernatant was labeled (45 min, 4 °C, 360 nm) with 0.5 µCi (0.25 µM) of [3H]BZDC-IP6
in the absence () or the presence (+) of a 1000-fold excess
IP4, IP6, or the unlabeled photoaffinity
analogues as competing ligands.
[View Larger Version of this Image (43K GIF file)]
Fig. 3.
Photoaffinity labeling of purified GST-Syt
II-C2B domain (10 µg, 5 µM). A, labeling
with [3H]BZDC-IP6 (0.4 µCi, 0.2 µM). This fluorogram was obtained following SDS-10%
polyacrylamide gel electrophoresis of purified GST-Syt II-C2B (1 µg)
irradiated (45 min, 4 °C, 360 nm) in the absence () or the
presence (+) of 50-, 250-, or 1000-fold molar excess of
IP3, IP4, or IP6 relative to the
concentration of photoaffinity label. B, relative
specificity of [3H]BZDC-IPn labeling of GST-Syt
II-C2B. Purified GST-Syt II-C2B was photoaffinity labeled with
P-1-tethered [3H]BZDC-IP3, P-1-tethered
[3H]BZDC-IP4, P-2-tethered
[3H]BZDC-IP6, and P-1-tethered
[3H]BZDC-IP6 (each, 0.4 µCi, 0.2 µM). The photolabeling was quantified using NIH IMAGE
1.59 and is expressed as a percentage of binding to P-2-tethered
[3H]BZDC-IP6. The asterisk
indicates that no labeling was observed with the P-1-tethered
[3H]BZDC-IP6 under these conditions.
[View Larger Version of this Image (22K GIF file)]
TTV
KKTLNPYFNESFSF346)
in this domain had been implicated in the binding of inositol polyphosphates to Syt II-C2B domain (9). Three central Lys residues
(underlined) were confirmed as crucial for IPn binding by
analysis of mutants containing one or more Lys to Gln substitutions
(10). However, it was not known whether a short peptide corresponding
to this central region would exhibit high affinity binding to
IPns. The 32-residue peptide was therefore obtained by solid
phase synthesis and purified to >95% purity by HPLC.
Fig. 4.
Competition between 32-mer peptide and
GST-Syt II-C2B for photoaffinity labeling by
[3H]BZDC-IP6. The lysine-rich 32-amino
acid peptide (0, 1, 2, and 4 molar equivalents as indicated
below the gel) was added to samples containing equimolar
amounts (2.5 µM) of purified GST-Syt II-C2B and
[3H]BZDC-IP6. The samples were preincubated
for 15 min at 0 °C and then photoaffinity labeled and
electrophoresed as described above to produce the fluorogram
shown.
[View Larger Version of this Image (11K GIF file)]
Fig. 5.
Circular dichroism spectra of 32-mer (50 µM in 145 mM phosphate-buffered saline, pH
7.4) at 11 °C, in the absence () and the presence of 0.1 (
),
0.5 (
), and 1.0 (
) molar equivalents of
IP6.
[View Larger Version of this Image (20K GIF file)]
Fig. 6.
Photoaffinity labeling of C2B domains of Syt
II and III with P-2-tethered [3H]BZDC-IP6.
A, diagram of the different GST fusion proteins employed for
labeling studies. B, alignment of the putative
IP6 binding regions of Syt II and Syt III-C2B domains. The
arrow indicates the splice site between Syt II and Syt III
sequences in the GST-Syt III/II-C2B chimera. C, fluorogram
showing the photoaffinity labeling of the purified GST-Syt C2 domain
fusion proteins (5 µM) with [3H]BZDC-IP6 (0.2 µM) in
phosphate buffer (pH 7.4). Samples were preincubated for 4 °C for 15 min, irradiated (360 nm, 0 °C, 45 min) and electrophoresed as
described above.
[View Larger Version of this Image (18K GIF file)]
C) construct showed modest but readily
detectable labeling (Fig. 6C). Essentially no covalent
modification was observed for the GST-Syt III/II-C2B chimeric protein,
perhaps attributable to the inaccessibility of the sterically bulky
BZDC moiety to the IP6 binding site of this chimera.
subunit of clathrin
assembly protein (AP-2) (32) had the highest affinity for
IP6, whereas the novel cytoskeletal linker protein
centaurin-
had the highest affinity for PIP3 (21). The
IP3 binding site of the affinity-purified rat brain
IP3 receptor (19) has been identified by sequencing a
proteolytic fragment of the [3H]BZDC-IP3
photoaffinity-labeled protein (33). The availability of two
regioisomeric-tethered derivatives of IP6 (23, 24) has
facilitated the further study of kinases (34, 35) and other proteins
that interact with IP6. The P-2-tethered
[3H]BZDC-IP6 prepared in this work was
employed to establish the location of the IP6 binding site
in the C2B region of the synaptotagmins and to confirm the ligand
specificity of this domain by an independent method. In the process,
the site specificity and ligand selectivity found fully validates the
use of the photoaffinity labeling approach in the search for and
active-site mapping of additional PIPn- and IPn-binding
proteins.
1 isoform can also be labeled
efficiently3 with
[3H]BZDC-IP3. Indeed, the C2B domain of Syt I
has been recently implicated in binding of PIP2 at high
calcium concentrations (37), providing further explanation for the
affinity of the photoaffinity probes for the Syt II-C2B domains
employed in this study.
, a novel multi-domain protein with
both homology regions, suggesting its role in communication of membrane
lipid changes to the cytoskeleton (21). Very recently, the N-terminal
region of the
subunit of AP-2 was found to have high affinity for
PIP3 (38), a finding foreshadowed by the highly selective
and efficient photoaffinity labeling of AP-2 by
[3H]BZDC-IP4 (31, 32). In unpublished work
from our laboratories,4
[3H]BZDC-IP4 showed efficient labeling of
assembly protein AP-3, already documented to have high affinity for the
higher IPns IP4, IP5, IP6,
and IP7 (39), which inhibited assembly of the clathrin
coat. Interestingly, PIP3 has now been shown to be more effective than the soluble IPns or other PIPns in
inhibiting AP-3-mediated clathrin coat
assembly.5 Finally, the labeling of
purified Syt II-C2B domains by [3H]BZDC-IP4
can be understood in terms of the affinity of Syt C2 domains for
PIP3 at low calcium concentrations (37).
C) showed
partial unmasking of the IPn binding domain.
*
This work was supported by National Institutes of Health
Grant NS 29632 (to G. D. P.) and in part by DuPont NEN. 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.
Present address: Dept. of Medicinal Chemistry, 308 Skaggs Hall,
College of Pharmacy, The University of Utah, Salt Lake City, UT 84112.
¶
Present address: Wyeth-Ayerst Research, 401 North Middleton
Rd., Pearl River, NY 10965.
Present address: Molecular Neurobiology Laboratory, Tsukuba
Life Science Center, Inst. of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan.
Supported by the Japanese Ministry of Education, Science, and
Culture.
§§
To whom correspondence should be addressed: Department of
Medicinal Chemistry, 308 Skaggs Hall, The University of Utah, Salt Lake
City, UT 84112. Tel.: 801-581-7063; Fax: 801-581-7087; E-mail: gprestwich{at}deans.pharm.utah.edu.
1
The abbreviations used are: Syt, synaptotagmin;
AP-2, assembly protein-2; BZDC, 4-benzoyldihydrocinnamoyl; GST,
glutathione S-transferase; Ins, inositol; IP3,
D-myo-Ins(1,4,5)P3; IP4,
D-myo-Ins(1,3,4,5)P4; IP6, Ins(1,2,3,4,5,6)P6; IPn, inositol
polyphosphate; PIP2, phosphatidylinositol
(4,5)-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PIPn, phosphoinositide polyphosphate; HPLC, high pressure liquid chromatography.
2
J. T. Elliott, unpublished results.
3
E. Tall, G. Dormán, P. Garcia, S. Shah, J. Chen, A. A. Profit, Q.-M. Gu, A. Chaudhary, G. D. Prestwich, and M. J. Rebecchi, submitted for publication.
4
A. A. Profit, J. M. Rabinovich, B. Mehrotra, G. D. Prestwich, and E. M. Lafer, unpublished observations.
5
W. Hao, Z. Tan, K. Prasad, K. K. Reddy, J. Chen,
G. D. Prestwich, J. R. Falck, S. B. Shears, and E. M. Lafer (1997)
J. Biol. Chem. 272, in press.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.