From the Department of Pediatrics, Division of
Clinical Chemistry and Biochemistry, University of Zürich,
CH-8032 Zürich, Switzerland and the § Department of
Biochemistry, University of Geneva,
CH-1211 Geneva 4, Switzerland
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ABSTRACT |
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The Ca2+-binding protein S100A2
is an unusual member of the S100 family, characterized by its nuclear
localization and down-regulated expression in tumorigenic cells. In
this study, we investigated the properties of human recombinant S100A2
(wtS100A2) and of two mutants in which the amino-terminal
Ca2+-binding site I (N mutant) and in addition the
carboxyl-terminal site II (NC mutant) were replaced by the canonical
loop (EF-site) of -parvalbumin. Size exclusion chromatography and
circular dichroism showed that, irrespective of the state of cation
binding, wtS100A2 and mutants are dimers and rich in
-helical
structure. Flow dialysis revealed that wtS100A2 binds four
Ca2+ atoms per dimer with pronounced positive
cooperativity. Both mutants also bind four Ca2+ atoms but
with a higher affinity than wtS100A2 and with negative cooperativity.
The binding of the first two Ca2+ ions to the N mutant
occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold
increase for the last two Ca2+ ions. A further 2-3-fold
increase of affinity was observed for respective binding steps of the
NC mutant. The Hummel-Dryer method demonstrated that the wild type and
mutants bind four Zn2+ atoms per dimer with similar
affinity. Fluorescence and difference spectrophotometry showed that the
binding of Ca2+ and Zn2+ induces considerable
conformational changes, mostly attributable to changes in the
microenvironment of Tyr76 located in site II. Fluorescence
enhancement of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid
clearly indicated that Ca2+ and Zn2+ binding
induce a hydrophobic patch at the surface of wtS100A2, which, as in
calmodulin, may be instrumental for the regulatory role of S100A2 in
the nucleus.
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INTRODUCTION |
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The S100 protein family constitutes a large subgroup of Ca2+-binding proteins of the EF-hand type and is composed of at least 16 different members displaying 25-65% identity at the amino acid level (1). These proteins contain two distinct EF-hand motifs, separated by a central hinge region. The carboxyl-terminal Ca2+-binding site (site II), called the canonical EF-hand, is composed of 12 amino acids and is common to all EF-hand proteins. The amino-terminal site (site I) contains 14 amino acids and is S100-specific. The three-dimensional structure of S100B (2, 3, 51) and S100A6 (4, 52) revealed a symmetric homodimeric fold that is unique among calcium-binding proteins. It has been suggested that the specificity of interaction with target proteins may be located in the central "hinge" region, which shows the lowest sequence homology among all S100 proteins (5, 6). Members of the S100 family interact with annexins (S100A6, -A10, and -A11); non-muscle tropomyosin and myosin (S100A4); intermediate filaments (S100A8 and -A9); and different enzymes, for example fructose-1,6-bisphosphate aldolase (S100A1 and S100B), guanylate cyclase (S100B), glycogen phosphorylase, adenylate cyclase (S100A1) (reviewed in Refs. 1, 7, and 8), giant myosin-associated twitchin kinase (9), and the SR Ca2+ release channel (both S100A1) (10).
Thirteen S100 genes were found to be localized in a cluster on human chromosome 1q21 (11-13). Contrary to calmodulin, the expression of S100 proteins is cell- and tissue-specific. A wide range of different human diseases has been associated with deregulated expression of S100 genes (1, 7, 14). S100 proteins have been implicated in pleiotropic cellular events, with specific functions for each of the family members, such as cell cycle regulation, cell growth, cell differentiation, and motility.
S100A2 (formerly called S100L) was first detected at high levels in a subset of cells in lung and kidney and moderately in liver, cardiac muscle, and skeletal muscle (15) and was later recognized as the product of a gene located on chromosome 1q21 (16). This protein is an unusual member of the S100 protein family because of its predominantly nuclear localization (17), compared with the cytosolic localization of the other S100 proteins. The cDNA coding for S100A2 was found through subtractive hybridization between normal and tumor-derived human mammary epithelial cells in a search for novel tumor suppressor genes (18). S100A2 is preferentially expressed in normal mammary epithelial cells but is down-regulated in breast tumor cells (19, 20) and in several other tumor types as well (17). Expression of S100A2 can be altered by the calcium ionophore A23187 (down-regulation) and by the demethylating agent 5-azacytosine (up-regulation) in cell culture (19). Investigations of the methylation status of the S100A2 promotor region in normal (HBL100) and tumorigenic (T47D) cell lines showed that S100A2 expression is suppressed by DNA methylation (21). In contrast, S100A4 is reported to be overexpressed in metastatic breast cancer cell lines and human tumor tissues (17). Similarly, the expression of S100A4 in mouse melanoma cells led to a metastatic phenotype (22). These results indicate that S100A2 and S100A4 are oppositely regulated and might have inverse functions in tumorigenesis. The protein S100A2 may play a role in inhibiting tumor initiation or suppressing tumor cell growth.
The relationship between primary structure and cation binding is poorly
understood not only in the S100 family but in general (23, 24).
Generally, the dimeric S100 proteins display a low affinity for
Ca2+ with a intrinsic association constant
(KCa') of about 3 × 103 M1 (for a review, see Ref.
8). These results were confirmed in more recent studies on S100A4,
S100A6, S100B, and S100A11 (25-27). Zn2+ can affect the
binding of Ca2+ to particular S100 proteins (28-30).
Indeed, using direct binding methods for S100B (27, 28), S100A4 and
S100A6 (25), S100A3 (31), S100A12 (32), and S100A9 (33), it was
demonstrated that they are able to bind Zn2+ with fairly
high affinity. Thus, S100 proteins have variable cation binding
properties in agreement with their diversified functions.
In order to elucidate the role of S100A2, we investigated the
Ca2+- and Zn2+-binding properties of this
nuclear protein under physiological conditions. We addressed the
question of how the binding of cations to S100A2 is dependent on the
intrinsic nature of the loops and on the cross-talk between the paired
sites I and II. What is the contribution of each of the sites to ion
binding and conformational properties? The approach was to characterize
ion binding and conformational properties of
wtS100A21 and two mutant
proteins, in which the loops of site I and site II were replaced by the
canonical loop (EF-site) of -PV. By difference spectrophotometry and
fluorimetry, we also compared the
Ca2+-dependent changes in the environment of
the Tyr residues. Finally, using bis-ANS fluorimetry, an exposure of
hydrophobic patches on the surface of the proteins was
demonstrated.
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EXPERIMENTAL PROCEDURES |
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Construction of Wild Type and Mutated Expression Vectors--
To
replace one or both of the functional Ca2+ N- or C-terminal
domain) of wtS100A2 (GenBankTM/EMBL accession number
M87068) by a canonical loop (EF-domain) of human -PV, the human
cDNA (GenBankTM/EMBL accession number X63070) was
amplified by PCR on Perkin-Elmer and Hybaid Omni-Gene thermal cyclers,
using specific oligonucleotides spanning the sequence of the EF-site of
PV and specific restriction sites for subsequent cloning into the
Bluescript SK+-vector (Stratagene).
Oligonucleotide Sequences-- Oligonucleotides were synthesized on a Gene Assembler DNA synthesizer (Pharmacia LKB). The primers used to amplify the cDNA were as follows: A2P1, 5'-GGTCTGCAGCAGATCCATGATGTGC-3'; A2P2, 5'-CATTTCGTCGACCCCAATTTTGCCGTCCCCATCTTTGTCGTACTTGTGGAAGGTAGGAC-3'; A2P3, 5'-ATTGGGGTCGACGAAATGAAGGAACTTCTGCACAAG-3'; A2P4, 5'-GGAGAATTCGTCAACCCCAATTTTGCCGTCCCCATCTTTGTCCAGGCTGCCCATCAGCTTCTT-3'; A2P5, 5'-GACGAATTCGCTGTTTTCCTGGCACTCATC-3'; A2P6, 5'-CAGGGATCCAAGAGTTCTGCTTCA-3'; A2P7, 5'-TGCAGCAGACATATGATGCAGT-3'. Underlined are the inserted restriction sites for BamHI, EcoRI, SalI, and PstI, and in italics is the sequence of the EF-site of PV. The resulting proteins are N mutant (change of positions 21-34) and NC mutant (change of positions 21-34 and 64-75).
Expression and Purification of Mutant Proteins-- Recombinant S100A2 proteins were expressed and purified as described previously (25). For the NC mutant, the purification procedure was modified in that the supernatant applied to the phenyl-Sepharose affinity column contained only 1 mM CaCl2, and proteins were eluted with 20 mM Tris-HCl (pH 7.5) containing 10 mM EGTA. The homogeneity of wtS100A2 and mutant proteins was examined by Tricine SDS-PAGE with 100 mM dithiothreitol (DTT) present in the protein samples.
Gel Electrophoresis and Mass Spectrometry-- Tricine SDS-PAGE (15%) was performed as described previously (34). For reducing conditions, 150 mM DTT was added to the sample buffer. Proteins were visualized by Coomassie Blue or silver staining. The molecular mass of the purified recombinant S100A2 proteins was analyzed by electrospray ionization mass spectrometry as described (25).
Western Blot Analysis--
Western blot analysis was performed
as described previously (17). The rabbit polyclonal antibodies against
human recombinant S100A2 and human recombinant -PV (F3866) were
diluted 1:5000 and 1:20,000, respectively. The protein bands were
visualized using alkaline phosphatase-conjugated secondary antibodies
(Promega) diluted 1:3000.
Metal Ion Determination and Metal Removal--
Removal of
contaminating metals, equilibration of the protein in the assay buffer,
and determination of total Ca2+ and Mg2+
concentrations were performed as described previously (35). The protein
concentration was determined from the ultraviolet absorption spectrum
using a molar extinction coefficient at 278 nm of 3030, 2920, and 1640 M1 cm
1 for wild type, N mutant,
and NC mutant monomer, respectively.
Secondary Structure Monitored by Circular Dichroism--
CD
spectra were acquired with a Jasco J-715 spectropolarimeter on
solutions of 0.25 mg/ml protein in 5 mM Tris-HCl buffer, pH
7.5, in a cell of 1-mm optical path. Ellipticities were normalized to
residue concentration using the relationship MRW =
o Mr/lc,
where
o represents the observed ellipticity in degrees,
Mr is the average molecular weight of an amino
acid in the polypeptide, l is the path length in mm, and
c the protein in g/liter.
Cation Binding-- Ca2+ binding was measured at 25 °C by the flow dialysis method (36) in 50 mM Tris-HCl, pH 7.5, 150 mM KCl (buffer A). Protein concentrations were 40-70 µM. Treatment of the raw data and evaluation of the metal binding constants were as described earlier (35). Since the Ca2+ binding isotherms showed either positive or negative cooperativity, the data were analyzed with the equation of Adair,
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(Eq. 1) |
Optical Methods to Probe the Environment of Tyr-- Emission fluorescence spectra were taken with a Perkin-Elmer LS-5B spectrofluorimeter on metal-free proteins at room temperature, with excitation and emission slits of 2.5 and 10 nm, respectively. The excitation wavelength was 278 nm. 25 µM EGTA or 5 mM CaCl2 or 0.5 mM ZnCl2 was added to obtain the metal-free, Ca2+ or Zn2+ forms, respectively. Denaturation curves were obtained by monitoring the Tyr fluorescence change upon titration of the metal-free proteins with guanidine-HCl and analyzed according to the method of Pace et al. (37).
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(Eq. 2) |
Titration of Exposed Hydrophobicity-- The Ca2+- and Mg2+-dependent changes in hydrophobic matrices of S100A2 or derivatives were followed by monitoring the fluorescence properties of 4,4'-dianilino-1,1'-binaphtyl-5,5'-disulfonic acid (bis-ANS) as described by Pan and Johnson (38). Solutions of 40 µM bis-ANS and 2 µM metal-free proteins (after the addition of 25 mM EGTA) were excited at 390 nm, and the emission spectrum was recorded with slits of 5 nm. 2 mM CaCl2 or 1 mM ZnCl2 was added to obtain the Ca2+ or Zn2+ form, respectively.
Protein Reduction and Thiol Reactivity-- The thiol reactivity was assayed after overnight incubation with 100 mM DTT at pH 8.5 followed by desalting on a Sephadex G-25 column equilibrated in nitrogen-saturated buffer A. The thiol reactivity was monitored by measuring the kinetics of the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or Ellman's reagent by spectrophotometry at 412 nm according to Riddles et al. (39).
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RESULTS |
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Expression, Purification, and General Properties of Recombinant
Human S100A2 Protein and Two Mutants--
S100A2 mutations were
designed to produce proteins with alterations in
Ca2+-binding sites I and/or II; the introduced amino acid
sequence changes are shown in Fig. 1. In
the N mutant, the 14-residue-long loop of site I was replaced by a
canonical 12-residue-long loop, corresponding to the sequence of the
EF-site of human -PV. To create the NC mutant, site II of S100A2 was
replaced by the same EF-site of PV (see "Experimental Procedures").
The NC mutant has an additional amino acid exchange, Y76F, due to the
EcoRI restriction site, which was introduced for the cloning
procedure. Both mutants should possess two functional HLH-type
Ca2+-binding sites. The cDNA coding for all S100A2
proteins was then cloned into the prokaryotic expression vector pGEMEX
for bacterial expression, giving rise to the construct pXA2 (wtS100A2),
pXN (N mutant), or pXNC (NC mutant). Extracts of bacterial cultures, before and after induction with
isopropyl-
-D-thiogalactopyranoside, were subjected
to SDS-PAGE (Fig. 2). Due to very low
expression, low solubility, and unusual biochemical properties, it was
not possible to purify sufficient amounts of the analogous C-mutant for
this study. The reason for this is still not known.
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Immunological Properties of wtS100A2 and Mutants--
The
immunological reactivity of the wild-type and mutant proteins with
polyclonal S100A2 and -PV antibodies was tested in Western blot
analyses in the presence of Ca2+ (Fig. 2, B and
C). With the polyclonal antibody against S100A2 (Fig.
2B), we observed a strong immunoreaction for the wtS100A2 monomer as well as for the dimer (lane 1).
Additionally, a weak signal of higher molecular weight was detected for
wtS100A2 (lane 1), probably representing a
trimeric form of the protein. In contrast, only faint signals were
detected with the mutants, especially with their dimeric forms
(lanes 2 and 3). This immunoreaction indicates that the amino acid exchanges modify the protein structure of
the mutants and therefore decrease the reactivity against the S100A2
antibody. The polyclonal antibody against human
-PV detected the
monomer and dimer bands of the N mutant and an additional band of ~16
kDa of unknown origin (Fig. 2C, lane
2). This band was barely visible by protein staining and may
represent the Ca2+ form of this mutant. In the case of the
NC mutant, only the monomers showed a signal with the antibody against
human PV (lane 3). The stronger immunoreaction
with the N mutant indicates that the additional EF-loop in the NC
decreases the immunoreactivity of this mutant. No reactivity was
observed against wtS100A2 (Fig. 2C, lane
1), indicating that the PV antibody recognizes the EF-site
of PV in the mutant proteins.
Biophysical Characterization of wtS100A2 and Mutants-- Size exclusion chromatography on a TSK125 gel filtration column (300 × 7.8 mm) equilibrated in buffer A under reducing conditions was carried out to determine if S100A2 and its mutant proteins existed as monomers or dimers in solution (data not shown). All protein forms eluted as single sharp peaks except for the Zn2+-loaded N mutant. Using the calibration mixture of protein standards from Bio-Rad, we found apparent molecular masses of 26-28 kDa, suggesting that they behave as dimers. This is in agreement with several other S100 proteins, even those that cannot form an intermolecular disulfide bridge. Zn2+-loaded N mutant showed an apparent molecular mass of 16 kDa, but the elution peak is broad and asymmetric, suggesting that this mutant partially dissociates into monomers upon binding of Zn2+.
Secondary Structure--
Fig. 3
shows the far UV CD spectra of the apo, Ca2+, and
Zn2+ forms of wtS100A2 and the two mutants. The spectra of
the Ca2+ and metal-free form of the wild type protein are
superimposable with an estimated -helix content of 50%.
Zn2+ binding causes a slight, but significant, decrease in
-helix content. The ellipticities of the N and NC mutants are about
1.3-fold lower than that of wtS100A2, suggesting that the
"normalization" of site I is at the expense of some
-helical
conformation or implies a simple change in the orientation of the
-helices (40). The ellipticity of these mutants is minimally
influenced by the binding of Ca2+ or Zn2+. In
summary, the secondary structure does not change significantly upon
binding of Ca2+ or Zn2+ to wtS100A2 or its
mutants. This feature is reminiscent of calbindin D9k, for
which the exclusive role as buffer was invoked as rationale (41).
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Direct Cation Binding Studies-- Ca2+ binding studies by flow dialysis revealed that the wtS100A2 dimer binds four Ca2+ with pronounced positive cooperativity (nH = 2.04) and a [Ca2+]0.5 of 470 µM (Fig. 4). The N and NC mutants also bind four Ca2+ per dimer but at independent sites of different affinities or sites with negative cooperativity. The binding isotherms were analyzed with the Adair equation assuming four sites with either pairwise identical affinities or with different affinities. The stoichiometric constants are summarized in Table II. Both models offer a reasonably good fit (thick and thin lines in Fig. 4), but for wtS100A2 the first model yields a calculated Hill coefficient of 1.87 with very discrepant values for the stoichiometric binding constants. This and the fact that the experimental value of the Hill coefficient is above 2 strongly suggest that the second model is more appropriated and that allosteric interactions occur between subunits of wtS100A2. Therefore, only the model of four different binding constants is further investigated in this study. In the N and NC mutants the low affinity sites have changed to high affinity sites, since binding of the first Ca2+ to the mutants occurs with a 100-400-fold higher affinity than in wtS100A2. In contrast, the second Ca2+ binds with 2-7-fold higher affinities in the N and NC mutant, respectively.
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Intrinsic Tyr Fluorescence-- wtS100A2 and the N and NC mutants each possess a Tyr20, adjacent to the amino terminus of the site I Ca2+-binding loop (Fig. 1). In addition, wtS100A2 and the N mutant possess a Tyr76 adjacent to the carboxyl terminus of the site II loop. The microenvironments of both Tyr residues may thus be strongly influenced by Ca2+ binding. Indeed, the metal-free forms of the three proteins show unusual fluorescence emission spectra, i.e. maxima at 320 nm that are blue-shifted to 310 in the presence of Ca2+ or Zn2+ (Fig. 6). In wtS100A2, cation binding is accompanied by an important (Zn2+) to moderate (Ca2+) enhancement of the fluorescence intensity. In the different native forms of the N mutant, the fluorescence intensities are lower but qualitatively similar to wtS100A2; they resemble those of S100A6, which possesses two Tyr in exactly the same positions. The fluorescence of the NC mutant yields low signals, and the spectra are qualitatively different from those of wtS100A2 and N mutant. This suggests that the environment of Tyr76 is much more perturbed by ion binding than Tyr20, the only probe in NC. They are similar to S100B, which also has one Tyr in the same position as the NC mutant. For all three proteins, the spectra in the presence of Mg2+ are the same as those of the apo forms (not shown), thus confirming the general conclusion that S100 proteins do not interact significantly with Mg2+. Positive cooperativity implies an allosteric conformational change after binding of the first Ca2+. A Ca2+ titration of the fluorescence change yielded a steep curve for wtS100A2 (Fig. 4, asterisks), slightly preceding the mean saturation curve.
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Near UV Difference Spectra-- The UV difference spectra (data not shown) of wtS100A2 show intense positive peaks at 280 and 288 nm (Fig. 7A) and are quite similar to those of S100A6 (25), which has an identical topography of Tyr residues. The difference spectra of the N mutant are similar to those of S100A2 but show 2-3-fold lower intensities, which corroborates the fluorescence data. The difference spectra of the NC mutant are small. Together, our data indicate that Tyr76, adjacent to the Z-position of site II, is highly responsive to cations, whereas the environment of Tyr20, located in the N-terminal helix of site I, is minimally perturbed by Ca2+ and Zn2+.
Bis-ANS Fluorescence Enhancement-- In polar solvents, the fluorescence of bis-ANS is weak, but it is strongly enhanced if the probe is associated with hydrophobic surface patches of proteins or other biomaterials (35). Fig. 8A shows how the different forms of wtS100A2 enhance the fluorescence of bis-ANS: 2-fold for the metal-free and Mg2+ form, 4- and 6-fold for the Ca2+ and Zn2+ forms, respectively. The N mutant also shows a 2-, 3- and 4-fold enhancement in the metal-free, Ca2+, and Zn2+ forms, respectively (Fig. 8B). These data clearly indicate that Ca2+ and Zn2+ induce a hydrophobic patch at the surface, which is qualitatively quite similar in the wild type and N mutant. The metal-free form of the NC mutant enhances the bis-ANS fluorescence 3-fold, Zn2+ binding does not lead to strong enhancement, and Ca2+ binding even leads to a slight decrease (Fig. 8C).
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Thiol Reactivity-- wtS100A2 contains four Cys residues per monomer in positions 3, 22, 87, and 94 (i.e. three outside the Ca2+-binding sites and one in site I), whereas the N and NC mutants lack Cys22 (Fig. 1). After thorough reduction and DTT removal as described by Föhr et al. (31), thiol titration with DTNB revealed that 2.8-3.0 thiols reacted rapidly in wtS100A2 (t1/2 < 10 s), 2.0-2.3 in N mutant, and 1.8-1.9 in NC mutant. The thiol reactivity is not affected by Ca2+, and the addition of 4 M guanidine-HCl did not uncover more thiols than could be titrated in the native proteins. It is likely that one Cys residue is very reactive and rapidly reoxidized to cystine after removal of the excess DTT, since during SDS-PAGE such disulfide-bridged covalent dimers are also observed (Fig. 2A and Ref. 14).
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DISCUSSION |
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S100A2 exerts its function in the cell nucleus by some presently unknown mechanism. Even in the dimeric state, S100A2 is small enough to enter the nucleoplasm via the nuclear pore complex by passive diffusion (for a review, see Ref. 43). Nuclear accumulation may occur if S100A2 is selectively retained through binding to a nondiffusible component in the nucleoplasm, thus offering a plausible explanation why S100A2 is predominantly found in the nucleus. The inner membrane of the nuclear envelope contains inositol 1,4,5-trisphosphate- and cADPR-modulated Ca2+-channels, which upon stimulation may raise the local Ca2+ to high levels. Nuclear and cytoplasmic Ca2+ signals in turn act on gene expression by coincidental activation of different components of the basic transcription machinery (44, 45). Hence, as a candidate tumor suppressor gene, S100A2 may well link the nuclear Ca2+ signal to one of the above mentioned transcription components. Indeed, some S100 members activate nuclear protein kinases (e.g. S100B activates the new Ndr kinase2 and binds to bHLH transcription factors (46, 47). The question arises as to whether S100A2 has the correct Ca2+ sensitivity and protein-interactive properties. The first purpose of this study was to evaluate these features. We expressed and purified the protein to electrophoretic homogeneity, verified the correct molecular weight, and observed, as for all other S100 members, that the protein is homodimeric, not merely by noncovalent interaction but also through formation of disulfide bridges. Usually, S100 proteins occur as dimers in vitro and in vivo, but S100A2 is also able to form higher polymers under nonreducing conditions (17).
In common with most S100 members, S100A2 binds four Ca2+
ions per protein dimer with moderate affinity but with much greater positive cooperativity than in S100A4, S100A6, S100A11, etc. Our study
does not allow us to identify the site to which Ca2+ binds
first or even to determine whether the binding to this allosteric unit
is sequential as in Nereis sarcoplasmic
Ca2+-binding protein SCP (48). But it is likely that site I
has a lower affinity than in other S100 members, since the sequence CQ
(underlined in Fig. 1) in the Ca2+-binding loop
of site I deviates strongly from the G(R/K) sequence encountered in the
other members. Our data indicate that the four Ca2+-binding
sites form an allosteric unit and that binding of the first two
Ca2+ ions overcomes an energetically expensive
conformational change and leads to a facilitated binding of the next
two Ca2+. wtS100A2 binds also two Zn2+ per
monomer with an affinity of 2 × 105
M1, i.e. 2-fold higher than in the
case of the unusual S100A3 (31). We have direct evidence that
Zn2+ does not bind to the same sites as Ca2+
and that, contrary to the case of some other S100 members,
Ca2+ and Zn2+ do not act synergistically but
rather in an antagonistic manner. Where are the two potential
Zn2+-binding sites located? The high affinity for
Zn2+ suggests that each binding site contains clusters of
at least two His and/or Cys residues (49). If human S100A2 has a
three-dimensional structure similar to S100A6 or S100B, two clusters
are observed per monomer: His18 + Cys22 + His40 and Cys3 + Cys87 + Cys93. Since our data showed that the thiols are well
exposed to the polar solvent, these may well represent potential
Zn2+-binding sites and would constitute a novel
Zn2+-binding motif.
Ca2+ and Zn2+ binding to S100A2 proceeds with minimal changes in the secondary structure but with marked changes in the environment of the two Tyr residues, as probed by fluorescence and near UV difference spectrophotometry. Bis-ANS fluorescence and phenyl-Sepharose chromatography indicate that Ca2+ binding also increases the surface-exposed hydrophobicity, which may render the protein more apt to interact with its potential targets in the nucleoplasm. Zn2+ binding shows similar conformational changes, but with different amplitudes, suggesting that Zn2+ also may promote interactions with different targets. If S100A2 acts as a trigger protein in the nucleus, one needs to know the local Ca2+ and Zn2+ concentrations in the nucleoplasm to evaluate the influence of these cations on the biological activity of S100A2. This information is presently not available, but, given the low affinity for Ca2+ and high affinity for Zn2+, it is likely that in vivo fluctuations of both cations trigger this protein's nuclear action.
The second purpose of this study was to evaluate the cross-talk between
the two Ca2+-binding sites of S100A2. Therefore, we
replaced the Ca2+-binding sites by the high affinity site
of -PV. In structure-function studies, it is important to confirm
that the mutations engineered into the parent protein do not
destabilize the general protein structure. A comparison of the CD
spectra, stability toward guanidine-HCl, and the quaternary structure
of wtS100A2 and the N mutant suggest that the mutation did not
dramatically affect the global folding, although local conformational
changes occur (see Tyr fluorescence), probably in the region involved
in hydrophobic interactions (see bis-ANS fluorescence). A very similar
loop grafting experiment on calbindin D9k also confirmed
that the overall structure remains the same (50). But the N-terminal
segment (4-6 residues) in the loop of site I, which is normally turned
"inside out" and coordinates Ca2+ by main chain
carbonyls, is in the mutant reoriented "outside out" as in
canonical EF-hands and coordinates Ca2+ with the side chain
carboxylates. A very similar phenomenon is expected to occur in the
case of S100 proteins, since the three-dimensional structure of the
Ca2+ forms of S100B (51-53) and S100A6 (54) shows that
site I has a conformation very similar to its conformation in calbindin
D9k.
The first mutation, replacement of the central loop of site I by that
of a canonical EF-hand, strongly increased K1
and K2 for Ca2+ while barely
modifying the K3 and K4
values, thus abolishing the positive cooperativity. The resulting
protein displays negative cooperativity or pairs of independent binding
sites of high and low affinity, respectively. The increase in affinity
of the first binding step may be explained in two ways: 1) the grafted
loop of -PV is intrinsically of high affinity (55, 56); 2) Fig. 8
suggests that the Ca2+-driven hydrophobic exposure, which
is energetically unfavorable, is much less extensive in the mutants
than in wtS100A2. Hence, binding energy is no longer sacrificed, and
the apparent affinity is higher. In the similar mutants in calbindin
D9k, the affinity of the mutated site for Ca2+
decreased 30-fold (57), since the wild-type protein shows no hydrophobic exposure, but the Ca2+ off-rate is increased in
the mutated "outside out" loop. The affinity for Zn2+
is only decreased by a factor of 4 by the replacement of site I; a
moderate change was also observed in the case of S100B (27) and
suggests that the EF-hands are not involved in Zn2+
binding.
When the central loops of both sites of S100A2 have been replaced by those of PV, including a replacement of Tyr76 by Phe, the CD spectra were still similar to those of wtS100A2, and the NC mutant had an even higher affinity for Ca2+ than the N mutant: K1' and K2' are increased 1.8- and 3.0-fold, respectively. The latter increase is significant and probably represents binding to site II. Again, the lesser, energetically unfavorable, conformational changes (Fig. 8, compare C and B) may explain why the affinity in the NC mutant is higher than in the N mutant.
In summary, the dimeric S100A2 displays an unusually low affinity for Ca2+ but very strong positive cooperativity. The latter is completely lost, but the Ca2+ affinity is strongly increased, in mutants with a canonical Ca2+-binding loop in site I. Two Zn2+ ions bind with an high affinity and induce, as Ca2+, conformational changes including an hydrophobic patch on the protein surface that may be instrumental for the interaction of S100A2 with nuclear targets.
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ACKNOWLEDGEMENTS |
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We thank Drs. Heinz Troxler and Peter Hunziker (University of Zurich) for performing the electrospray ionization mass spectrometry analysis, Udo Redweik for amino acid analysis, Irène Fasso for circular dichroic experiments, and Margrith Killen for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Swiss National Science Foundation Projects 31-50510.97 and 31-37575.93; EMDO-Stiftung; Julius Klaus-Stiftung; Union Bank of Switzerland (on behalf of a client); Biomed-2 (BBW number 95.0215-1, Switzerland); and European Union Grant BMH4-CT96-0319.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. Fax: 041-22-7026483; E-mail: jos.cox{at}biochem.unige.ch.
1 The abbreviations used are: wtS100A2, recombinant human S100A2 expressed in E. coli; N mutant, S100A2 with the EF-loop of parvalbumin in site I; NC mutant, S100A2 with the EF-loop of parvalbumin in both sites I and II; PV, parvalbumin; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DTT, dithiothreitol; bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); GdnHCl, guanidinium HCl.
2 B. W. Schäfer, and C. W. Heizmann, unpublished data.
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