From the Department of Biochemistry and McLaughlin
Macromolecular Structure Facility, University of Western Ontario,
London, Ontario N6A 5C1, Canada and the Departments of
§ Environmental Health, ¶ Emergency Medicine, and
Cell Biology, Anatomy and Neurobiology, University of Cincinnati
College of Medicine, Cincinnati, Ohio 45267-0056
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ABSTRACT |
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The calcium-binding protein S100B (an S100 dimer
composed of two S100 The S100s are a group of proteins that belong to the EF-hand
calcium-binding protein family (1-3). This family includes such mechanistically well understood proteins as the
calcium-dependent muscle sensor troponin-C, the ubiquitous
enzyme regulator calmodulin, and the visual signaling molecule
recoverin. Signaling by these molecules is controlled through calcium
binding to the EF-hand protein and subsequent induction of a
conformational change that modifies protein-protein interactions with a
target protein. For example, troponin-C undergoes a calcium-induced
conformational change, allowing a strengthening of its interactions
with a second member of the troponin complex, troponin-I (4, 5). An
analogous mechanism has been proposed for several S100 proteins,
allowing them to control such diverse processes as protein
phosphorylation, cytoskeletal protein assembly, neurite outgrowth, and
cell cycle regulation through a variety of calcium-sensitive
interactions with other proteins (3, 6, 7).
S100B (an S100 dimer composed of two S100 In order to clarify potential target proteins, a bacteriophage random
peptide library has recently been used to define a recognition sequence
for S100B (18). These studies showed that a 12-residue sequence
containing the consensus motif (K/R)(L/I)XWXXIL
was sufficient to bind to S100B in a calcium-sensitive manner. Further
studies have shown this peptide (TRTK-12) successfully competes with
other proteins such as glial fibrillary acidic protein and CapZ for calcium-sensitive S100B binding (10, 18). Similar approaches have
identified several 15-residue sequences, analogous to the sequences for
myosin light chain kinase, melittin, and mastoparan, as representative
samples of the calcium-dependent target proteins for
calmodulin (19). A clear distinction exists between these binding
"epitopes" for S100B and calmodulin as the sequences of TRTK-12 and
the calmodulin peptides are not related by alignment. However there is
a similarity in composition in that both peptides have a preponderance
of hydrophobic and basic residues.
Recently, NMR spectroscopy and x-ray crystallography have been used to
determine the three-dimensional structures of calcium-saturated human
(20), rat (21), and bovine S100B (22). The structures revealed the
S100B dimer has two symmetric monomers each comprising two EF-hand
calcium binding sites. The N-terminal EF-hand is formed from helices I
and II where helix I (and I' from the other monomer) are integral to
the maintenance of the dimer. Likewise the C-terminal calcium binding
site (site II) is composed of helices III and IV where helix IV (and
IV') interact at the dimer interface. Calcium binding to S100B has a
minimal effect on the conformation of the N-terminal site I but has a
pronounced effect on the canonical C-terminal EF-hand. This results in
a reorientation of helix III with respect to other helices in the
protein (20-23). Further, it has been observed that a hydrophobic
region composed of several residues near the C terminus of helix IV and
in the linker between sites I and II is present on the surface of
Ca2+-S100B1 (20).
Based on this structural data, these regions have been proposed to form
a possible recognition site for calcium-sensitive protein-protein
interactions in S100B. The composition of this site, primarily
hydrophobic and acidic residues, is reminiscent of the protein
recognition surface in calmodulin (17). In addition, the amino acid
sequences in the C-terminal helix and linker show the least sequence
similarity among the S100 protein family, suggesting a rationale for
target protein specificity (3, 24, 25).
Several groups have reported that some of the S100 proteins including
S100B, S100A12, S100A6, S100A11, calgranulin C, and S100A3
are not only calcium-binding proteins but also bind Zn2+
with high affinity (26-30). Further, zinc binding has the pronounced effect of increasing calcium affinity in S100B (26) and calgranulin C
(29) by at least 10-fold. Such an observation is unique in the EF-hand
calcium-binding protein family and may indicate a new mode for calcium
regulation and signaling in the S100 proteins. The impact of these
observations was recently demonstrated for the giant protein kinase
twitchin, where the addition of Zn2+ to calcium-bound
S100A12 increased the kinase activation by more than
30-fold over that with calcium alone (16). With this in mind, the
current work studies the interaction of the 12-residue peptide TRTK-12
with S100B as a function of the divalent metal ions Mg2+,
Ca2+, and Zn2+ in order to understand the
influence of each metal ion on TRTK-12 binding. We have used the S100
protein S100A11 and an N-terminal peptide from S100B to determine
whether, at least in this case, TRTK-12 binding is specific for S100B.
Further, we have used NMR spectroscopy and the three-dimensional
structure of human Ca2+-S100B to highlight residues that
may be important for TRTK-12 interaction.
Materials--
[15N]Phenylalanine,
[15N]alanine, Tris-d11,
CH3CO2Na-d3, and
deuterium oxide were obtained from Isotec Inc. (Miamisburg, OH). Calcium chloride, magnesium chloride, and zinc chloride were all puratronic grade from Alfa-Aesar (Mississauga, Canada). All other chemicals used were of the highest purity commercially available. The
auxotrophic strain DL39 AvtA::Tn5 (31) was kindly donated by
Dr. L. McIntosh (University of British Columbia).
Recombinant human S100B was expressed in Escherichia coli
(strain N99) and purified to homogeneity as described previously (32).
The backbone amides of alanine and phenylalanine residues of S100B were
selectively 15N-labeled and purified as described
previously (33). TRTK-12 (TRTKIDWNKILS) peptide was synthesized and
purified as reported (18). Bacterially expressed and purified S100A11
(34) was a kind gift of Dr. Michael Walsh (University of Calgary,
Calgary, Alberta, Canada). The hs1bI peptide (residues 1-46) from
human S100B (SELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEE) was custom synthesized by Chiron Mimotopes (Clayton, Victoria, Australia) using
Fmoc (N-(9-fluorenyl)methoxycarbonyl)chemistry and purified by reversed-phase high pressure liquid chromatography.
Fluorescence Spectroscopy--
Spectra were obtained using a
Hitachi F-4010 fluorescence spectrophotometer equipped with a stirred
cell holder. Tryptophan fluorescence was excited at 295 nm and
emission-scanned from 305 to 450 nm using an emission band pass of 5 nm. Titrations of the TRTK-12 peptide with S100B were followed by
monitoring the increase in fluorescence at 332.8 nm. The concentrations
of the TRTK-12 peptide and S100B stock solutions were determined from
absorbance spectra using extinction coefficients of NMR Spectroscopy--
All NMR spectra were acquired on a Varian
Unity 500 MHz spectrometer equipped with a triple-resonance,
pulsed-field gradient probe. Carrier frequencies used were centered at
120.0 (15N) and 4.73 (1H) ppm. One-dimensional
1H NMR spectra for TRTK-12 were collected at 25 °C.
TRTK-12 (~1 mg) was dissolved in 0.5 ml of 20 mM
CD3CO2Na, 50 mM KCl, pH 6.5. Typically, a spectral width of 6000 Hz was used with the transmitter set on the H2O resonance. Water suppression was
accomplished during a 2.0-s relaxation delay between transients using a
weak presaturation pulse. All spectra were referenced to the
trimethylsilyl resonance of sodium
2,2-dimethyl-2-silapentane-5-sulfonate at 0.00 ppm, zero-filled to
65,536 points, and processed using line broadening of 0.5 Hz.
Two-dimensional 1H-15N HSQC experiments were
acquired on a 0.5 mM S100B sample at 35 °C using the
sensitivity-enhanced method (35) as described previously (33).
The calcium-binding protein S100B has been suggested to interact
with a variety of cellular targets in a calcium-sensitive fashion.
Rather than utilizing a speculative target, we have used a synthetic
peptide, TRTK-12, to probe the calcium and zinc-sensitive binding to
S100B. With several three-dimensional structures of S100B now in hand,
we have used this data to identify potential sites for TRTK-12
interaction with Ca2+-S100B and correlate this with the
identification of a potential biological target.
Interaction of TRTK-12 with S100B--
The intrinsic fluorescence
spectrum of TRTK-12 is shown in Fig.
1A. The peptide displays an
emission maximum at 354 nm for the single Trp7 in the
sequence. This wavelength of emission is consistent with the tryptophan
located in a polar aqueous environment. For interaction with S100B,
this tryptophan provided a convenient marker, since S100B itself
contains no tryptophan residues. The addition of two equivalents of
apo-S100B to TRTK-12 resulted in a minimal change in the TRTK-12
tryptophan fluorescence, indicating that any interaction between the
apo-S100B calcium-binding protein and TRTK-12 is very weak. The
addition of physiological levels of Mg2+ (5 mM)
to the apo-S100B/TRTK-12 solution had little effect on the tryptophan
fluorescence of TRTK-12 despite previous observations that
Mg2+ may bind to S100B (36). In contrast, the addition of
saturating amounts of calcium to the apo-S100B/TRTK-12 solution
resulted in a 40% enhancement and a blue shift to 332.8 nm for
tryptophan fluorescence. These observations are consistent with
previous results showing that TRTK-12 is able to interact with the
calcium form of S100B (18, 37). Further, the blue shift indicates the
tryptophan residue moves to a more nonpolar environment. S100B has been
shown to tightly bind 2 mol of Zn2+ per monomer, and
binding of this metal causes a significant change in the surface
hydrophobicity of the protein (26). As shown in Fig. 1A,
binding of Zn2+ to apo-S100B resulted in little enhancement
or shift of the TRTK-12 fluorescence. These observations indicate that
calcium is the primary metal responsible for TRTK-12 binding to
S100B.
To determine whether TRTK-12 fluorescence and binding to
Ca2+-S100B could be enhanced or reduced by other metal
ions, we examined the additional influences of Mg2+ and
Zn2+ on tryptophan fluorescence (Fig. 1B). In
the presence of Ca2+-S100B the addition of 5 mM
Mg2+ had a small negative effect on the fluorescence
intensity of TRTK-12. This finding was similar to the observed small
effect in Fig. 1A, where the addition of Mg2+
alone had only a weak effect on Trp7 fluorescence. In
contrast, the addition of Zn2+ to the
Ca2+-S100B solution resulted in an approximate 10%
increase in tryptophan fluorescence with no further change in the
emission maximum. These results are consistent with Zn2+
binding to S100B and enhancement of the interaction of TRTK-12 with
Ca2+-S100B. The addition of 5 mM
Mg2+ to this sample resulted in a small decrease in
tryptophan fluorescence similar to that observed in the absence of
Zn2+.
While the above work and other studies have indicated that TRTK-12
interacts with S100B in a calcium-sensitive manner, the specificity of
TRTK-12 for other S100 proteins has not been examined. We investigated
this using an S100 protein that has some sequence differences from
S100B, especially in the linker and C-terminal regions. S100A11 is an
S100 protein (also called S100C) originally isolated from cardiac
muscle (34), which, like S100B, has been shown to bind both
Ca2+ and Zn2+ (38). Fig. 1C shows
the tryptophan fluorescence spectra obtained for the addition of
Ca2+-S100B and Ca2+-S100A11 to a TRTK-12
solution. The spectra show the characteristic shift in TRTK-12
fluorescence in the presence of Ca2+-S100B. However, the
addition of Ca2+-S100A11 yielded little change in the
TRTK-12 fluorescence emission wavelength or amplitude, indicating that
Ca2+-S100A11 does not perturb TRTK-12 fluorescence. This
result is consistent with little or no interaction between TRTK-12 and
Ca2+-S100A11.
In an effort to localize the region of Ca2+-S100B that
interacts with TRTK-12, we studied the effect of a 46-residue peptide comprising a single EF-hand from the N-terminal region of S100B (hs1bI)
on TRTK-12 fluorescence. This peptide has been shown to be mostly
TRTK-12 Affinity for S100B--
The interaction of TRTK-12 with
Ca2+-S100B was measured by following the tryptophan
fluorescence emission at 332.8 nm. Since the previous section showed
that TRTK-12 does not interact with apo-S100B, it was important to
ensure that Ca2+-S100B was the major populated species
during these titrations. To address this, two titrations of TRTK-12
with S100B were done in the presence of differing amounts of excess
calcium (1 and 10 mM). Under these conditions and given the
calcium dissociation for S100B (~7-200 µM) (40),
Ca2+-S100B should be the dominant species. Thus, the
interaction of TRTK-12 should not be affected by the apo-S100B to
Ca2+-S100B equilibrium. Fig.
2 shows three representative titrations of TRTK-12 with Ca2+-S100B. Fig. 2 shows that TRTK-12
fluorescence at 332.8 nm increases as a function of added
Ca2+-S100B, a result of TRTK-12 binding to
Ca2+-S100B. There is excellent agreement between the two
curves, indicating that Ca2+-S100B must be the predominant
form of the protein at both calcium concentrations and that the
interaction of TRTK-12 with Ca2+-S100B is the major event
being monitored by these titrations. The shape of the curves does not
reveal the stoichiometry of TRTK-12 binding to Ca2+-S100B,
which has been suggested to be either 1 or 2 molecules of TRTK-12/S100B
dimer (i.e. 1 molecule of TRTK-12/dimer or 1 molecule of
TRTK-12 for each S100
Fluorescence experiments shown in Fig. 1 indicated that tryptophan
emission of TRTK-12 was enhanced in the presence of both Zn2+ and Ca2+ compared with Ca2+
only. To determine whether this was a direct result of increased affinity caused by Zn2+, titrations to determine the
binding affinity of TRTK-12 for Ca2+-S100B were done in the
presence of Zn2+. As with the previous calcium titration
experiments, a Zn2+ concentration of 20-fold the S100 Regions of TRTK-12 Affected by Binding to S100B--
The residues
particularly influenced by TRTK-12 binding to Ca2+-S100B
were examined by 1H NMR spectroscopy. Fig.
3 shows the aromatic, tryptophan indole NH, and methyl regions of a series of 1H NMR spectra of
TRTK-12 with increasing amounts of Ca2+-S100B added. The
complete assignment of TRTK-12 1H resonances, in the
absence of S100B, was accomplished using standard two-dimensional
methods (data not shown). In the absence of Ca2+-S100B, the
resonances for TRTK-12 were sharp, with 1H chemical shifts
close to those representative of a random coil structure. As the
concentration of Ca2+-S100B was increased, several of the
resonances in TRTK-12 shift and broaden dramatically, while others are
less affected. For example the indole NH of Trp7 in TRTK-12
broadens and shifts downfield by 0.25 ppm. In addition, this resonance
becomes similar in line width to those observed for other amide
resonances in Ca2+-S100B, indicating that this residue now
has similar relaxation properties as the Ca2+-S100B
protein. The magnitude of the shift of the Trp7 indole NH
indicates that the koff for the
TRTK-12·Ca2+-S100B complex is on the order of 125 s Regions of S100B Affected by TRTK-12
Binding--
Fluorescence studies showed that the TRTK-12
tryptophan fluorescence was affected very little in the presence
of apo-S100B. This was reinforced by 1H-15N
HSQC spectra of apo-S100B in the absence and presence of TRTK-12, which
indicated the backbone resonances of apo-S100B are not influenced by
equimolar amounts of TRTK (data not shown). Qualitatively, this would
indicate that the affinity of TRTK-12 for apo-S100B is
conservatively 10-fold larger than the apo-S100B
concentration (1 mM S100
In previous work, we determined that calcium binding to S100B results
in excessive line broadening, making studies of the Ca2+-S100B species more difficult than apo-S100B. We had
shown that this line broadening was a result of aggregation of
Ca2+-S100B in the absence of a target protein such as
TRTK-12 (32). To reduce the possibility of Ca2+-S100B
aggregation in this work, we studied the interaction of TRTK-12 with
Ca2+-S100B by the addition of incremental amounts of
Ca2+ to apo-S100B/TRTK-12 solutions. This yielded identical
results compared with direct TRTK-12 addition to
Ca2+-S100B. As shown in Fig.
4A, the
1H-15N HSQC spectrum of Ca2+-S100B
and Ca2+-S100B/TRTK-12 show some significant differences.
In particular, the largest changes in the N and NH chemical shifts
(weighted average >0.25 ppm) were noted for residues
Asp12, Ser41, Phe43,
Ile47, Val53, Val56,
Thr59, Asp61, Ala78,
Ala83, Cys84, Phe87,
Phe88, and His90. In the case of alanine and
phenylalanine residues, these assignments were confirmed using a
specifically 15N-labeled sample (Fig. 4B).
Interestingly, we observed little change in the position of
Ile11 upon binding of TRTK-12. This was consistent with
observations for binding of a p53 peptide to S100B (41) but different
from a previous study of TRTK-12 binding to bovine S100B, where
Ile11 was observed to undergo the largest shift of any
residue (37).
The focus of this work was to determine how the interaction of the
TRTK-12 binding epitope for S100B varies with metal-ion binding and
whether this binding showed a degree of specificity for S100B. These
aspects of S100B interaction have not been dealt with in previous
studies with TRTK-12. In addition, we have used our data to probe a
region on the Ca2+-S100B three-dimensional structure that
may be responsible for peptide interaction.
The affinity of TRTK-12 for Ca2+-S100B is approximately 1 µM. This value is higher than previously measured in the
absence of added salts (0.15 µM) and may reflect a
sensitivity of peptide binding to ionic strength which is known to have
a significant negative influence on S100B calcium affinity. The
magnitude of the TRTK-12 dissociation constant is consistent with those
found for other calcium-binding protein-peptide complexes such as TnI peptides binding to skeletal muscle troponin C (24 µM)
(4) and caldesmon peptides binding to calmodulin (1 µM) (42, 43). The interaction of TRTK-12 with S100B is
Ca2+-specific, since neither Mg2+ nor
Zn2+ binding to S100B could stimulate peptide binding. The
finding that Zn2+ binding alone to S100B is unable to
promote TRTK-12 binding is in contrast to results using the hydrophobic
probe TNS, where a large increase in TNS fluorescence is observed in
the presence of Zn2+-S100B (26, 27, 44). This observation
occurs as a result of the interaction of TNS with the Zn2+
form of S100B. Since TNS is known to be a probe for hydrophobic surface
rather than a specific binding site, these differences indicate that
the hydrophobic surface exposed in S100B by Zn2+ binding is
not specific for TRTK-12 binding. In turn, this probably indicates that
the protein does not adopt a proper conformation in the
Zn2+ form to allow target protein binding.
The most dramatic effect on peptide binding to S100B is in the presence
of both Zn2+ and Ca2+, where an increase in
peptide affinity of about 5-fold is noted compared with the presence of
Ca2+ only. This observation is consistent with results for
S100A12, where a 30-fold increase in twitchin kinase
activity was observed upon the addition of Zn2+ to the
calcium form of the protein (16). Together with previous observations
that S100B is able to bind two Zn2+ ions per monomer (26,
44) this indicates that binding of TRTK-12 is enhanced by
Zn2+ binding to S100B in the presence of Ca2+
only. Since TRTK-12 does not appear to bind to Zn2+-S100B
as judged by the present experiments, it would appear the calcium
binding to S100B is critical. This indicates that Zn2+
plays more of a structural role in proteins such as S100B and S100A12 rather than a regulatory role. Similar proposals
have been suggested for the S100 proteins S100A3 (30) and calgranulin C
(29). Consistent with this idea, the affinity of Zn2+ for
S100B (10 The measured affinity of TRTK-12 for Ca2+-S100B represents
at least a 5-fold increase in affinity compared with that for a
23-residue peptide from the tumor suppressor protein, p53 (residues
367-388) (41). Further, Zn2+ binding to S100B did not have
a notable effect on p53 peptide binding. These observations indicate
there are some clear differences between these two peptide species. As
indicated by Wilder et al. (15), the p53 peptide studied
previously fits some of the TRTK-12 consensus sequence but lacks
residues Ile10 and Leu11. Further, sequence
analysis of nine unique p53 sequences from the PIR data base reveals
that all forms of p53 do not resemble the TRTK-12 binding motif for
these last two residues.2
This may explain the decreased affinity of p53 for S100B. It is
intriguing that secondary structure prediction methods show that
TRTK-12 should form an amphipathic The results presented in this work allow an initial overview of the
TRTK-12 interaction with Ca2+-S100B previously attempted in
the absence of a Ca2+-S100B three-dimensional structure
(37). Residues in the N terminus (Asp12), linker and N
terminus of helix III (Ser41, Phe43,
Ile47, Val53), and C terminus
(Ala78, Ala83, Cys84 and
Phe88) undergo the most significant changes in chemical
shift upon TRTK-12 binding. Since TRTK-12 has little interaction with
the N-terminal peptide, hs1bI, from S100B this would indicate that the
linker and C terminus are more important. Using the structure-activity relationship (46), it can be proposed that residues change chemical shift as a direct result of interaction with TRTK-12. Fig.
5 shows the structure-activity
relationship for Ca2+-S100B based on changes in chemical
shift. The S100B structure clearly shows these residues comprise a
large localized hydrophobic surface on the protein. Further, several
residues including Phe43, Ala83,
Phe87, and Phe88 are included in or near this
region and increase their accessible surface area more than 20% upon
calcium binding (20). These observations are consistent with a region
for interaction of TRTK-12 with S100B.
monomers) is proposed to act as a
calcium-sensory protein through interactions with a variety of
proteins. While the nature of the exact targets for S100B has yet to be
defined, random bacteriophage peptide mapping experiments have
elucidated a calcium-sensitive "epitope" (TRTK-12) for S100B
recognition. In this work, interactions of TRTK-12 with S100B have been
shown to be calcium-sensitive. In addition, the interactions are
enhanced by zinc binding to S100B, resulting in an approximate 5-fold
decrease in the TRTK-12/S100B dissociation constant. Moreover,
Zn2+ binding alone has little effect. TRTK-12 showed
little evidence for binding to another S100 protein, S100A11 or to a
peptide derived from the N terminus of S100B, indicating both a level
of specificity for TRTK-12 recognition by S100B and that the N-terminal
region of S100B is probably not involved in protein-protein
interactions. NMR spectroscopy revealed residues most responsive to
TRTK-12 binding that could be mapped to the surface of the
three-dimensional structure of calcium-saturated S100B, revealing a
common region indicative of a binding site.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
monomers) is one member of
the S100 protein family for which several potential cellular targets
have been identified. For example, polymerization of cellular
architecture molecules such as glial fibrillary acidic protein and
tubulin can be inhibited through a calcium-dependent interaction with S100B (8-10). Alternatively, S100B can inhibit the
phosphorylation of proteins such as myristoylated alanine-rich C kinase
substrate (11), the Alzheimer protein Tau (12, 13), and p53 (14, 15)
through interaction with these proteins rather than with the kinase
responsible. This action is different from that of the S100 protein
S100A12, which has been shown to inhibit phosphorylation of
the myosin protein kinase twitchin through interaction with its
regulatory region (16) in a fashion reminiscent of calmodulin's action
with myosin light chain kinase (17).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
280 = 5600 cm
1 M
1 for TRTK-12 and
280 = 3400 cm
1
M
1 for S100B. Samples of TRTK-12 were
typically made in 50 mM KCl, 50 mM Tris buffer
at pH 7.2. Additions of S100B or hs1bI were made using 1-2-µl
volumes of the proteins in 50 mM KCl, 50 mM Tris buffer at pH 7.2 using a calibrated Hamilton 10-µl syringe. Total sample volumes did not change by more than 3%.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Tryptophan fluorescence spectra of 1.05 µM TRTK-12 in 50 mM Tris, 50 mM
KCl at pH 7.2. A shows TRTK-12 alone (a),
and with 1.02 µM apo-S100 (b), 1.02 µM S100
and 5 mM MgCl2
(c), 1.02 µM S100
and 1 mM
CaCl2 (d), and 1.02 µM S100
and
20.4 µM ZnCl2 (e). B
shows TRTK-12 with 1.02 µM S100
and 1 mM
CaCl2 (a) and with 20.4 µM
ZnCl2 (b), 5 mM MgCl2
(c), or 20.4 µM ZnCl2 and 5 mM MgCl2 (d). C shows
TRTK-12 alone (a) and with 1.02 µM S100
and
1 mM CaCl2 (b) or with 1.18 µM S100A11 and 1 mM CaCl2
(c). D shows TRTK-12 alone (a) and
with 1.02 µM S100
and 1 mM
CaCl2 (b) or 1.0 µM hs1bI and 1 mM CaCl2 (c). In all cases,
fluorescence is expressed in relative units, and background buffer has
been subtracted.
-helical by circular dichroism spectroscopy, similar to S100B, and
is able to form a tetramer analogous in structure to the arrangement of
the four EF-hand motifs in the S100B dimer (39). As shown in Fig.
1D, the addition of hs1bI in the presence of calcium
resulted in little change in TRTK-12 fluorescence compared with TRTK-12
alone. This observation indicates that the N terminus of S100B alone is
not sufficient to interact with TRTK-12.
monomer) (37). Indeed, fitting of the above
data for either a single TRTK-12 or two TRTK-12 molecules binding to
Ca2+-S100B yielded very similar results. However,
examination of titration data in the presence of Zn2+ or as
monitored by NMR spectroscopy (see below) clearly indicated a
stoichiometry of 1 TRTK-12 molecule/Ca2+-S100
monomer. A
further titration was done using a 40% more dilute TRTK-12 solution.
As expected, the change in fluorescence was correspondingly lower, and
a subtler hyperbolic curve was obtained. From these data, a
dissociation constant of 0.91 ± 0.17 µM for TRTK-12
binding to Ca2+-S100B was determined.
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Fig. 2.
Titration of TRTK-12 with S100B in 50 mM Tris, 50 mM KCl at pH 7.2. The change
in fluorescence ( F), monitored at 332.8 nm, is plotted as
a function of the S100
/TRTK-12 ratio for 1.02 µM
TRTK-12 peptide in the presence of 1 mM CaCl2
(
), 1.02 µM TRTK-12 peptide with 10 mM
CaCl2 (
), 1.02 µM TRTK-12 peptide with 1 mM CaCl2 plus 20-fold ZnCl2
(compared with [S100
] (
), and 0.58 µM TRTK-12
with 1 mM CaCl2 (
).
concentration was chosen based on the reported dissociation constants
of S100B for Zn2+. The data plotted in Fig. 2 show that
Zn2+ binding to S100B in addition to Ca2+
increased the response of TRTK-12 toward S100B. From these data, a
stoichiometry of 1:1 TRTK-12:S100
monomer is clearly evident. Iterative curve fitting of this data yielded a dissociation constant of
0.18 ± 0.01 for TRTK-12 binding to
Zn2+/Ca2+-S100B, an approximate 5-fold tighter
binding than to Ca2+-S100B alone.
1. In the methyl region, Ile5 from TRTK-12
experiences similar line broadening as Trp7 but has a 0.3 ppm upfield chemical shift change. The figure also indicates that line
broadening is not consistent for all residues in the TRTK-12 peptide.
An interesting observation is the differential changes in
Thr1 and Thr3
-CH3 groups when
Ca2+-S100B is added. In the early additions of
Ca2+-S100B, the
-CH3 group of
Thr3 is broadened more so than that of Thr1.
Together, these observations provide evidence that the TRTK-12 peptide
is binding to Ca2+-S100B.
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Fig. 3.
Series of 500-MHz 1H NMR spectra
showing the effects of Ca2+-S100 on TRTK-12
peptide. The lower spectrum shows 1.1 mM TRTK-12 in
90% H2O, 10% D2O with 20 mM
CH3CO2Na-d3, 50 mM KCl, pH 6.5, at 25 °C. Assignments of the TRTK-12
resonances were done using standard two-dimensional methods and are
indicated. The sample was titrated with Ca2+-S100
,
giving the Ca2+-S100
/TRTK-12 ratios shown at the
right to a final concentration of 1.2 mM
S100
, 3.08 mM Ca2+ for 1.1 mM
TRTK-12 peptide. The dotted lines represent
resonances for Trp7
, Thr3
-CH3, and Ile5
-CH3, which
shift and broaden as a function of added
Ca2+-S100
.
).
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Fig. 4.
1H-15N HSQC spectra
of uniformly and selectively labeled 15N-phenylalanine and
15N-alanine recombinant 15N-labeled human S100B
showing changes in resonance position as a function of added TRTK-12
peptide. A shows spectra of 1 mM uniformly
15N-labeled Ca2+-S100 in the absence
(light contours) and presence (darker
contours) of 1 mM TRTK-12 peptide, respectively,
in 10 mM Tris-d11, and 50 mM KCl in 90% H2O/10% D2O, pH
7.26. The arrows are used to show resonances, which shift by
greater than 0.25 ppm (
|(1H)| + 0.2 *
|(15N)|) upon the addition of TRTK-12 peptide.
B shows resonances from phenylalanine and alanine residues
in 1.0 mM selectively 15N-labeled S100
with
1.0 mM TRTK-12 in the absence (light
contours) and presence of 2.0 mM
Ca2+ (darker contours). The spectrum
of apo-S100
is identical to that obtained in the absence of calcium
(33). The arrows show the change in resonance position upon
the addition of calcium resulting from both calcium binding
to S100
and binding of TRTK-12.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
7 M) is near the physiological
intracellular concentrations of free Zn2+, which are
thought to be in the micromolar range. It has been previously proposed
that Zn2+ can act as a structural ion in other S100
proteins based on the observation of a
His-X3,4-His pattern found near the C termini (30). This pattern is similar to that observed for
Zn2+-catalytic sites that frequently display a
His-X3-His motif (45). It is intriguing to note
that the three-dimensional structure of human Ca2+-S100B
shows that one possible Zn2+ binding site might include
residues His85 and His90 based on their
proximity in the structure (20). This region ultimately appears to be
important for peptide binding, since several changes in chemical shift
and exposed surface area are noted for residues in the C terminus.
-helix between residues Lys4 and Leu11. If this structure exists in the
S100B-protein complex, it would place residues Thr3,
Trp7, Leu10, and Ile11 on one side
of the helix, allowing integral contact of Leu10 and
Ile11 with S100B. On the surface, this would provide
differences between the interactions of TRTK-12 with
Ca2+-S100B compared with p53.
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Fig. 5.
Surface representation of
Ca2+-S100 showing the regions of greatest change in
chemical shift upon the addition of TRTK-12. Chemical shift
changes were plotted for residues where the weighted average of
1H and 15N shift (
|(1H)| + 0.2 *
|(15N)|) changed more than 0.15 ppm
(yellow) and 0.25 ppm (red) upon the addition of
TRTK-12. The surface was generated using InsightII (MSI) for the
coordinates of human S100B (20).
S100B has been proposed to interact with a large number of potential
target proteins including the head domain of glial fibrillary acidic
protein that bears some sequence similarity to TRTK-12 (10). Most
recently, studies have probed this interaction with p53 (15, 41),
guanylate cyclase, and p80 (47, 48). During the course of the current
work, a series of synthetic peptides were used to map the regions of
S100B that stimulate guanylate cyclase activity and phosphorylation of
p80. In agreement with our current findings, these studies indicated
that the C-terminal region of S100B encompassing residues
Thr81-Glu91 was most effective for guanylate
cyclase activation. It was also apparent that residues
Leu32-Leu40 could elicit a similar response.
This region is just N-terminal to residue Ser41 observed
here. Interestingly, our peptide hs1bI (residues 1-46) does not bind
to TRTK-12, perhaps suggesting a weaker contribution from the linker
region when TRTK-12 is bound and a stronger contribution for guanylate
cyclase. Supporting this possibility, it has also been noted that
guanylate cyclase does not contain a region similar in sequence to
TRTK-12, so this may indicate a fine difference between interaction of
S100B and its target proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. J. Dixon (Department of Physiology, University of Western Ontario) for the use of his fluorescence spectrophotometer. Funding for the NMR spectrometer in the McLaughlin Macromolecular Structure Facility was made possible through grants from the Medical Research Council of Canada and the Academic Development Fund of The University of Western Ontario and generous gifts from the R. Samuel McLaughlin Foundation and London Life Insurance Co. of Canada.
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FOOTNOTES |
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* This work was supported by a grant from the Medical Research Council of Canada (to G. S. S.).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 addressed. Tel.: 519-661-4021; Fax: 519-661-3175; E-mail: shaw{at}serena.biochem.uwo.ca.
The abbreviations used are:
Ca2+-S100B, S100, and -S100A11, calcium-saturated S100B,
S100
, and S100A11, respectively; HSQC, heteronuclear single quantum
coherence spectroscopy.
2 K. A. McClintock and G. S. Shaw, unpublished results.
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REFERENCES |
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