©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Cation Binding Properties of the Recombinant Human S100 Calcium-binding Protein A3, an EF-hand Motif Protein with High Affinity for Zinc (*)

(Received for publication, March 31, 1995; and in revised form, June 26, 1995)

Ursula G. Föhr (1) Claus W. Heizmann (1)(§) Dieter Engelkamp (1) Beat W. Schäfer (1) Jos A. Cox (2)

From the  (1)Department of Pediatrics, Division of Clinical Chemistry, University of Zurich, CH-8032 Zurich, Switzerland and (2)Department of Biochemistry, University of Geneva, CH-1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The calcium-binding protein S100A3 is an unusual member of the S100 family, characterized by its very high content of Cys. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified the recombinant human protein in Escherichia coli. The recombinant protein forms noncovalent homodimers, tetramers, and polymers in vitro with a subunit molecular weight of 11,712. The Zn-binding parameters of S100A3 were studied by equilibrium gel filtration and yielded a stoichiometry of four Zn per monomer with a [Zn](0.5) of 11 µM and a Hill coefficient of 1.4 at physiological ionic strength. The affinity for Ca is too low to be determined by direct methods (K > 10 mM). Ca- and Zn-binding can be followed by optical methods: the Trp-45 fluorescence is high in the metal-free form and addition of Zn and Ca, but not of Mg, leads to a 4-fold quenching. Ca and Zn promote also quite similar conformational changes in the Tyr and Trp environment as monitored by difference spectrophotometry. Fluorescence titrations with Zn confirmed that there is one set of high affinity binding sites with a [Zn](0.5) of 8 µM and a Hill coefficient of 1.3. Binding of Zn to a second set of low affinity sites induces protein precipitation. Fluorescence titrations with Ca confirmed the very low affinity of S100A3 for this ion with a [Ca](0.5) of 30 mM and slight negative cooperativity. Mg has no effect on this binding curve. Of the 10 Cys residues in S100A3, 5 only are free thiols, and accessible to 5,5`-dithiobis(2-nitrobenzoic acid); they display a high reactivity in the metal-free and Ca form, but a 20-fold lowered reactivity in the Zn form of S100A3. Ca-binding promotes the formation of a solvent-accessible hydrophobic surface as monitored by the 60-fold fluorescence increase of 2-p-toluidinylnaphthalene-6-sulfonate, whereas Zn has no noticeable influence. Our data indicate that Ca and Zn do not bind to the same sites and that under physiological conditions S100A3 is a Zn-binding rather than a Ca-binding protein; nevertheless, very specific conformational changes are introduced by either Ca or Zn. Since no Zn-binding motif of known structure was identified in the primary sequence of S100A3, the results are suggestive for a novel Zn-binding motif.


INTRODUCTION

The S100 protein family constitutes a subgroup of Ca-binding proteins of the EF-hand type displaying 30% or more sequence identity (Kligman and Hilt, 1988; Hilt and Kligman, 1991). Under physiological conditions their affinity for Ca is rather low but can be increased once S100 proteins are associated with their targets. Different S100 proteins were also found to bind Zn with a fairly high affinity (Baudier et al., 1986; Leung et al., 1987; Filipek et al., 1990; Dell'Angelica et al., 1994). Both intracellular roles, such as activation of enzymes, regulation of motility, and smooth muscle contraction, and extracellular roles, such as neuronal differentiation, glial proliferation, and prolactin secretion (for review, see Donato(1991), Zimmer and Dubuisson(1993), and Heizmann and Braun(1995)), have been proposed. Intriguingly, in different cases where calmodulin was thought to be the regulatory CaBP, (^1)S100 proteins were finally the real activators (Bianchi et al., 1993). Most S100 proteins interact in vitro with hydrophobic matrices, with membranes, enzymes, cytoskeletal and contractile proteins, and even cell surface receptors (for review, see Donato(1991)). All of these data point to a multifunctional role of the S100 family with a particular function for each of its members. This functional specificity is supported by the fact that their expression is differentially deregulated in different types of cancer cells (Hilt and Kligman, 1991; Weterman et al., 1992; Davis et al., 1993; Pedrocchi et al., 1994a, 1994b), suggesting participation in tumor progression. However, for none of these putative functions have the molecular details been elucidated.

The protein S100A3, (^2)formerly called S100E, was recognized for the first time as the product of one of the tightest gene clusters discovered in the human genome located on chromosome 1q21 (Engelkamp et al., 1993). The S100A3 gene shows a low but general transcription level in diaphragm, heart, skeletal muscle, stomach, lung, liver, fat tissue, and placenta. A YAC clone from human chromosome 1q21 has been recently isolated on which nine different genes coding for S100 proteins were localized. The clustered organization of S100 genes in the 1q21 region allowed to introduce a new logical nomenclature for these genes (Schäfer et al., 1995). The S100A3 gene product is 101 residues long and possesses one S100-type noncanonical Ca-binding loop of 14 residues expanding from Ala-20 to Glu-33, and one canonical EF-hand loop of 12 residues from Asp-63 to Glu-74, both flanked by two alpha-helices. In calbindin D-9k, the prototype of this S100 protein family with a resolved three-dimensional structure (Szebenyi and Moffat, 1986; Carlström and Chazin, 1993), the alpha-helices are oriented in an antiparallel fashion, thus forming a 4-helix barrel. Within the S100 subfamily S100A3 is unique for the exceptionally high number of Cys residues. Despite the Cys frequency, S100A3 does not display the classical zinc-binding motifs seen in metallothioneins (Vallee and Auld, 1990), DNA-binding proteins (Pérez-Alvarado et al., 1994), or protein kinase C (Hommel et al., 1994).

In order to begin to understand the role of S100A3 and the molecular mechanisms by which it exerts its function, we characterized in this study the Ca- and Zn-binding properties of recombinant human S100A3 under physiological conditions. We monitored the cation-dependent changes in the environment of the Trp and Tyr residues, probed the thiol/disulfide state and the cation-dependent reactivity of the thiols, and finally monitored the solvent-exposed hydrophobic surface. The results suggest that under physiological conditions S100A3 is a Zn-binding rather than a Ca-binding protein.


EXPERIMENTAL PROCEDURES

Materials

A protein fusion and expression system was obtained from New England Biolabs. Isopropyl-thio-beta-D-galactopyranoside and restriction endonucleases were from Boehringer Mannheim. Concentrated T4 DNA ligase was obtained from New England Biolabs. EDTA, ampicillin, and lysozyme were purchased from Fluka. Hydroxylapatite (Bio-Gel HTP), polyacrylamide, and electrophoresis equipment were from Bio-Rad.

Oligonucleotides were synthesized on a Gene Assembler DNA synthesizer (Pharmacia Biotech Inc.). The primers used to amplify S100A3 cDNA for cloning into pMal-c2 were as follows: S100A3-M, 5`-ATGGCCAGGCCTCTGGAGCAGG-3`; S100A3-B, 5`-GGCAAGTCCAGATTGAAAGGGG-3`.

Cloning of Human S100A3 into a Prokaryotic Expression System

Human cDNA of S100A3 was amplified by the polymerase chain reaction (PCR) as described earlier (Engelkamp et al., 1993), using the primers S100A3-M and S100A3-B. The resulting PCR product comprised the complete coding region of S100A3 beginning with the starting codon ATG. The PCR product was blunt end ligated into the XmnI-digested expression vector pMal-c2 downstream the malE gene, which encodes maltose-binding protein (MBP). Colonies of transformed Escherichia coli TB1 strains expressing MBP-S100A3 fusion protein were analyzed for their correct DNA sequence inserted into the expression vector by the dideoxynucleotide chain termination method.

Expression and Purification of Recombinant S100A3

Luria-Bertani medium (1000 ml) containing 0.2% glucose and 100 mg/liter ampicillin was inoculated with a 10-ml overnight culture of an E. coli TB1 clone expressing MBP-S100A3 fusion protein. At an A of 0.5, 1 mM isopropyl-thio-beta-D-galactopyranoside was added to induce the expression of the fusion protein. The culture was further incubated for 3 h. Cells were harvested by centrifugation at 4000 g (20 min, 4 °C), resuspended in a 50-ml cold column buffer (20 mM Tris/HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM NaN(3)) and frozen at -20 °C overnight. Cells were lyzed by thawing, incubation with 1 mg/ml lysozyme for 20 min on ice, and sonication for 1.5 min (on ice). This suspension was centrifuged at 9000 g (30 min, 4 °C). The crude extract (supernatant) was diluted to 2.5 mg/ml and applied to an amylose resin affinity column (length, 8 cm; diameter, 2.5 cm). The fusion protein was eluted as described by the manufacturer's manual (New England Biolabs) and incubated with 0.2% (by mass) protease factor Xa for 2-4 days at room temperature. The protease cleavage site is located exactly in front of the amino-terminal methionine of S100A3. The completeness of the cleavage was controlled by SDS-Tricine-PAGE (Fig. 1). S100A3 was separated from MBP by ion exchange chromatography followed by a second amylose resin affinity chromatography. Purity and concentration of recombinant S100A3 was controlled by SDS-Tricine-PAGE (Fig. 1) and amino acid analysis.


Figure 1: Expression and purification of recombinant human S100A3. Coomassie Blue-stained 15% SDS-Tricine-PAGE under reducing conditions, showing induction and purification steps of MBP-S100A3 fusion protein and of S100A3. Lane 1, 40 µg of crude extract of E. coli; lane 2, 40 µg of flow-through following loading onto amylose-resin column; lane 3, 5 µg of eluate from amylose-resin column: MBP-S100A3 fusion protein; lane 4, 25 µg of fusion protein after factor Xa cleavage; lane 5, 3 µg of MBP; lane 6, 2.5 µg of finally purified S100A3 (monomer).



One- and Two-dimensional PAGE and Molecular Weight Determination

15% SDS-Tricine gels were cast and run according to the method described by Schägger and von Jagow(1987). For two-dimensional PAGE, the precast Immobiline gels with an immobilized pH gradient from pH 3 to 10.5 (Promega) were used under denaturing conditions according to the producer's instructions.

The native apparent molecular weight of the metal-free, Ca and Zn forms of S100A3 was determined by gel filtration on a 1 70-cm column of Sephadex G-75 in 50 mM Tris buffer, pH 7.5, 150 mM KCl, 1 mM dithiothreitol (buffer A) containing either no divalent cations, 100 mM CaCl(2), or 100 µM ZnCl(2). The column was standardized with the calibration mixture of Bio-Rad.

Mass Spectrometry

Electrospray ionization mass spectra were obtained with a Sciex Api III and a Finnigan TSQ 700 instrument equipped with an ion-spray source. The protein molecular mass was determined from the acquired spectra with ESI deconvolution software from Finnigan.

Amino Acid Analysis

Amino acid analysis was performed by gas-phase HCl hydrolysis, conversion with dansyl chloride and subsequent evaluation of the derivatized amino acid products with a Beckman System Gold HPLC instrument.

Direct Zn-binding Studies

For removal of contaminating metal ions, S100A3 was precipitated with 3% trichloroacetic acid and then passed through a 1 40 cm Sephadex G-25 column equilibrated in the assay buffer. The protein concentration was determined from the UV absorption spectrum using a molar extinction coefficient at 280 nm of 14,500 M cm for metal-free S100A3. These values were measured on protein stock solutions in bidistilled water whose concentrations were determined by quantitative amino acid analyses.

Zn binding was measured at room temperature by the equilibrium gel filtration method of Hummel and Dryer(1962). A Sephadex G-25 column (0.7 50 cm) was equilibrated in buffer A containing variable concentrations of Zn. 0.5-1 ml of 50-200 µM metal-free protein was applied to the column. In the eluant Zn concentrations were determined by atomic absorption with a Perkin-Elmer 2380 atomic absorption spectrophotometer. For the atomic absorption measurements EDTA up to 1 mM was added to all solutions, including the standards (Titrisol, Merck). Protein concentrations were measured by ultraviolet absorption.

Optical Methods to Probe the Environment of Aromatic Residues

Emission fluorescence spectra were taken with a Perkin-Elmer LS-5B spectrofluorimeter. The measurements were carried out on 1 µM trichloroacetic acid-treated metal-free S100A3 at room temperature with excitation and emission slits of 5 nm. The excitation wavelength was 280 nm. 100 mM CaCl(2), 30 mM MgCl(2), or 200 µM ZnCl(2) was added to obtain the metal-free, Ca, Mg, and Zn forms, respectively. The denatured form was obtained by addition of 4 M guanidine-HCl. Ca and Zn titrations were done with 2 µM S100A3 in buffer A.

UV absorption spectra and difference spectra were measured with a Perkin-Elmer Corporation 5 UV/VIS spectrophotometer. Difference spectra were taken at room temperature on solutions with an optical density at 280 nm close to 1.

Protein Reduction and Thiol Reactivity

The influence of cations on the thiol reactivity was assayed on S100A3 samples that were previously reduced by overnight incubation with 100 mM DTT at pH 8.5 and chromatographed on a Sephadex G-25 column (0.7 35 cm) equilibrated in nitrogen-saturated buffer A. The thiol reactivity was assayed by monitoring spectrophotometrically at 412 nm the kinetics of the reduction of Ellman's reagent according to Riddles et al.(1983). The reaction was initiated upon mixing the protein solution with 10 µl of DTNB to a final concentration of 0.3 mM. Titration of Exposed Hydrophobicity-The Ca- and Mg-dependent changes in hydrophobic matrices of S100A3 was followed by monitoring the fluorescence properties of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as described by McClure and Edelman(1966). Solutions of 10 µM metal-free S100A3 and 0.5 µM TNS were exited at 328 nm and the emission spectrum recorded with slits of 10 nm.


RESULTS

Expression and Purification of Recombinant S100A3

To express and isolate S100A3 in large amounts, human S100A3 cDNA was cloned into the prokaryotic expression vector pMal-c2. A PCR product of human S100A3 cDNA was introduced at the protease factor Xa cleavage site behind the malE gene of pMal-c2 to generate a MBP-S100A3 fusion construct. The amount of expressed MBP-S100A3 fusion protein corresponded to about 12% of total cell protein of a bacterial culture. After isolating the fusion protein by amylose resin affinity chromatography S100A3 was cleaved off from MBP by the protease factor Xa. S100A3 was finally purified by ion exchange chromatography and a second amylose resin affinity chromatography. The correct cleavage of the fusion protein and the purity and concentration of S100A3 was controlled by amino acid analysis and SDS gel electrophoresis. Fig. 1shows the expression and purification of the fusion protein and of S100A3.

Biochemical Properties of Human Recombinant S100A

After classical treatments of S100A3, such as dialysis, ultrafiltration, and freezing, the S100A3 protein is partly insoluble, but dissolves quickly and completely when solid DTT up to 50 mM is added. SDS-PAGE clearly shows that different disulfide-linked oligomers are formed, which are reduced to the monomer after DTT treatment (data not shown).

To verify recombinant S100A3 for correct synthesis in bacteria we determined its exact mass by electrospray ionization mass spectrometry. Before desalting with butyl-300 microbore reversed-phase HPLC it was again necessary to mix the protein probe with 50 mM DTT to prevent precipitation on the column and to obtain any mass signal. In acidic solvent a molecular weight of 11,712 ± 1.7 was obtained, which is in good agreement with the calculated molecular weight, including the amino-terminal methionine, of 11,713.3 (Fig. 2). This result shows the correct expression of S100A3 in E. coli TB1, including an unprocessed amino-terminal methionine.


Figure 2: ESI-MS of human recombinant S100A3. ESI-MS data for S100A3 including 50 mM DTT.



SDS-PAGE after reduction of S100A3 with 10 mM DTT for 30 min at 37 °C also yielded a band with a molecular mass of 11 kDa. Determination of the apparent molecular mass by gel filtration on Sephadex G-75 after thorough reduction yielded different values depending on the presence of divalent cations: 22.4 kDa in the absence of divalent cations, 24.9 kDa in the presence of 100 µM Zn, and 39.0 kDa in the presence of 100 mM Ca. Thus, as in other members of the S100 family, S100A3 forms a noncovalent homodimer. Moreover, 100 mM Ca promotes formation of a higher order oligomer (likely tetramers). It is not clear if the latter phenomenon is to be attributed to the specific binding of Ca or to an ionic strength effect, which is known to stabilize hydrophobic interactions.

We assessed the isoelectric point of S100A3 by two-dimensional gel electrophoresis under reducing conditions using an immobilized pH gradient ranging from pH 3.5 to 10. In contrast to the calculated pI of 4.53, the determined pI of the denatured protein was found to be 5.5 (data not shown). This divergence may be caused by the experimental conditions and not by any modifications of the protein as the measured mass of recombinant S100A3 was found to be identical to the calculated value.

Direct Cation Binding Studies

Trichloroacetic acid can be used to remove ions, concentrate the protein, and prepare the sample correctly for Hummel-Dryer experiments. Gel filtration on Sephadex G-25 in 350 µM free Ca shows very little binding (<0.2 mol of Ca/mol of S100A3), indicating that a binding study of this cation cannot be carried out by direct means. Zn binding by the Hummel-Dryer method (Fig. 3) yields an isotherm of which the maximum value is somewhat difficult to evaluate since the protein shows a tendency to aggregate above 100 µM free Zn. The Scatchard plot, although curved upward indicating positive cooperativity, allowed a rather precise extrapolation to four binding sites per monomer (not shown). Assuming a maximal binding of 4 Zn per monomer, the Hill plot was calculated and yielded a Hill coefficient (n(H)) of 1.4 and a [Zn](0.5) of 11 µM (inset). A Hummel-Dryer experiment in the presence of both Ca and Zn indicated that there is neither competition between the cations nor reenforcement of affinity, as was described for S100A1 (Leung et al., 1987) or calgranulin C (Dell'Angelica et al., 1994).


Figure 3: Zn binding to S100A3 as determined by the Hummel-Dryer method. The solid lines are the theoretical isotherms calculated with the Hill equation with [Zn](0.5) = 11 µM and n(H) = 1.4. Inset, Hill plot of the data assuming four binding sites per monomer.



Fluorescence Characteristics

Fluorescence spectra (Fig. 4) indicate that denatured S100A3 has a fluorescence maximum 10-fold lower than that of the metal-free form. In the latter form and in the presence of Mg the Trp is very well shielded with (max) at 340 nm. Mg does not affect the spectrum, suggesting that no binding occurs. Ca, Zn, or Co binding leads to a 3-fold fluorescence decrease, in the case of Ca with a 7-nm blue shift, in the case of Zn or Co without any shift. Given the very good signal change, Ca, Zn, and Co titrations could be carried out by fluorimetry on a 2 µM solution of S100A3. Since S100A3 displays a very low affinity for Ca, the ratio of bound to added Ca is negligible. For Ca a smoothly increasing sigmoid was observed (Fig. 5A), indicating more than one site with different affinities or displaying negative cooperativity. This isotherm can be analyzed with [Ca](0.5) of 35 mM and n(H) equal to 0.76 (Fig. 5B). The same isotherm was obtained in 30 mM Mg, indicating that the sites are specific. For Zn there are two levels of signal change (Fig. 5A), one with a midpoint of 8 µM and one at 600 µM. The high affinity compartment, with four Zn binding sites (see above), shows pronounced positive cooperativity with n(H) equal 1.3 as analyzed in Fig. 5C. Zn binding to the lower affinity compartment (at 200-1000 µM free Zn) induces protein aggregation, as evidenced from strongly increased signals at emission wavelengths(285-290) close to the excitation wavelength (280 nm). Addition of both Ca and Zn leads to much stronger protein precipitation. Co binding seems monophasic with a K of 1 mM (data not shown). Apparently this Co-binding compartment corresponds to the high affinity compartment of Zn. Difference spectrophotometry on the S100A3 Co complex in the 240-800 nm zone shows that the complex does not display the peaks in the 650-750 zone, which are so characteristic for classical zinc fingers. Thus, the absence of these bands and the comparable low affinities for Zn and Co (nM for Zn and µM for Co in zinc fingers) suggest that in S100A3 there is no such motif.


Figure 4: Tryptophan fluorescence characteristics of S100A3 after excitation at 280 nm. Emission spectrum in the absence of metals (bulletbulletbulletbulletbullet), in the presence of 30 mM MgCl(2)(- - - - -), of 150 mM CaCl(2) (--), of 200 µM ZnCl(2) (-bullet-bullet-bullet-bullet), and of 4 M guanidine-HCl (thin dotted line). The protein concentration was 1 µM. The spectra were corrected for the buffer contribution.




Figure 5: A, conformational titration of 2 µM S100A3 with Ca in the presence (up triangle, filled) and absence () of 30 mM MgCl(2) and with Zn in the absence of other divalent cations (bullet) as followed by changes in the Trp fluorescence. The solid line is the Hill equation with [Ca](0.5) = 35 mM and n(H) = 0.76 and assuming a total signal change of 1.18. Panels B and C represent the Scatchard plot of Ca binding and of the high affinity component of Zn binding, respectively.



Difference Spectrophotometry

Difference spectra (Fig. 6) yielded rather small signal changes (10-20% of what is usually found in CaBPs), which could be essentially attributed to changes in the Tyr, to a lesser extent also to the single Trp environment. Qualitatively the spectra resemble very much those of the calmodulin-like protein (Durussel et al., 1993), with the appearance of negative peaks at 279 and 289 nm. They point to the shift of Tyr from a hydrophobic to a polar environment. The Phe environment seems not to be sensitive to cation binding. The difference spectra of the Zn and Ca forms are quite similar, although only Ca binding provoked the broad negative peak, likely due to a change in the polarity of the Trp environment (Ilich et al., 1988). Zn concentrations above 100 µM lead to progressive precipitation of the protein, resulting in a declining base line. Strongly enhanced precipitation is observed when millimolar concentrations of Ca were added to these Zn-containing samples.


Figure 6: Difference spectra of S100A3 (66 µM) in buffer A at room temperature after addition of 100 mM Ca (--) or 190 µM Zn(- - - - -) to the metal-free protein. The difference in optical density was expressed for a protein solution with an optical density of 1.0 at 280 nm.



Changes in the Thiol Reactivity Induced by Zn

Thiol titration with DTNB on a freshly reduced (250 mM DTT for 24 h at room temperature, followed by Sephadex G-25 chromatography) sample yielded 4.5-4.9 free thiols per protein monomer. Addition of 4 M guanidine hydrochloride increased these values to 5.0 and 5.3. This, together with the results of SDS-PAGE and mass spectrometry, indicates that S100A3 contains two to three intrapolypeptide disulfide bridges. The reactivity of the free thiols in metal-free and Ca form of S100A3 (Fig. 7) is similar and too high for measurement by classical means. Zn binding decreases the reactivity of these thiols by a factor of 20 and here again the presence of Ca does not noticeably affect the reactivity. The kinetic profiles in the presence of Zn do not obey to a Guggenheim equation for a pseudo-first-order rate reaction (Durussel et al., 1993). Either the 5 thiols have from the beginning a different rate constant, or the progressive blocking of thiols by bulky DTNB leads to steric hindrance. The presence of Ca does in no case modify the kinetics suggesting that the Zn-sensitive thiols are located on the opposite end of the EF-hands.


Figure 7: Thiol reactivity in S100A3 as monitored by the absorbance at 412 nm after addition of DTNB. Metal-free S100A3 (bulletbulletbulletbulletbullet); S100A3 in the presence of 200 mM Ca (--), 100 µM Zn(- - - - -), and 200 mM Ca + 100 µM Zn (-bullet-bullet-bullet-bullet). Protein concentration was 6.7 µM. After 15 min of reaction time 31 µM thiols were titrated. The reactions do not follow pseudo first-order kinetics.



Interaction with the Hydrophobic Probe TNS

Upon binding of Ca, CaBPs of the activator type, such as calmodulin (Tanaka and Hidaka, 1980) and neuron-specific CaBPs (Cox et al., 1994), usually display one or more solvent-exposed hydrophobic patches on their surface, which can be monitored with particular fluorescent probes such as TNS. Fig. 8shows that Ca binding induces a 30-fold increase in fluorescence enhancement, whereas no enhancement at all is observed upon binding of Zn. It should be noted that the development of the Ca-induced hydrophobic patch(es) occurs in a biphasic manner: one phase is very rapid (occurs within the time of mixing) and may well present the exposure of hydrophobic residues in each monomer; the second phase occurs over a range of 10s of minutes and may correspond to the transition of the dimer to the 38-kDa oligomer as shown by gel filtration. It should be noted that addition of even 220 mM Ca does not lead to noticeable aggregation, as monitored by turbidimetry (not shown). But in the presence of both 55 mM Ca and 95 µM Zn the enhancement is half of that of Ca alone, indicating that the binding is noncompetitive. Very similar results have been obtained with the fluorescent probe 1-anilinonaphtalene-8-sulfonate (data not shown).


Figure 8: Hydrophobic exposure in S100A3 as monitored by the fluorescence of TNS after excitation at 328 nm. Protein and TNS concentrations were 5 and 0.5 µM, respectively. TNS alone (thin solid line); metal-free S100A3 (bulletbulletbulletbulletbullet); S100A3 + 180 mM Ca (--); S100A3 + 190 µM Zn(- - - - -); S100A3 + 180 mM Ca + 86.5 µM Zn (-bullet-bullet-bullet-bullet); S100A3 + 190 µM Zn+ 90 mM Ca (thin dotted line).




DISCUSSION

In this study we report the biochemical characterization and cation-binding properties of S100A3, a new member of the S100 family with an unusually high content of Cys residues. The protein is a dimer and contains two EF-hand motifs per monomer. But, whereas most other S100 proteins display Ca dissociation constants of 0.1-1 mM, S100A3 is able to bind Ca only in the 10-100 mM free Ca range, i.e. very far from the cytosolic Ca levels. Nevertheless, this binding seems specific since it is accompanied by Tyr and Trp conformational changes very similar to those caused by Zn binding, by a well defined exposure of hydrophobicity and an oligomerization. The reason for this low affinity for Ca is not clear, since its primary structure is quite classical for a S100 member. However, our data indicate that each dimer contains five disulfide bridges. This may stabilize the protein but can impose strong constraints for the efficient binding of Ca. Reduction of all the disulfide bridges of S100A3 under denaturing conditions and alkylation of the thiols yields a protein product which binds Ca with a dissociation constant of 0.8 mM, (^3)i.e. an affinity close to that of most other S100 proteins. It is still possible that S100A3 displays a real Ca-dependent function when associated with its target or when secreted in the Ca-rich extracellular fluid. In contrast to calgranulin C (Dell'Angelica et al., 1994) and S100B (Baudier et al., 1986), there are no indications that Zn increases the affinity of S100A3 for Ca. But an interesting dynamic regulation may occur through zinc binding, since the affinity is rather high (K 10 µM). It is estimated that 99% of the 36 mg of Zn per kg human wet weight is intracellular and 25% of this amount is not, or loosely bound (Vallee and Falchuk, 1993). This represents intracellular free Zn concentrations of 40-400 µM, depending on the tissue, with a maximum of 2 mM in the retina. It is thus very likely that in vivo S100A3 is mostly in Zn-bound form. Its precise role in the direct activation of response systems and/or in promotion of exchange with other important Zn-regulated proteins must still be evaluated.

Where are the four Zn sites per monomer, or rather the eight Zn sites per native dimer located and what kind of sequence motifs in S100A3 could be responsible for Zn binding? Since the three-dimensional structure of most S100 proteins has not been elucidated, one can only compare with Zn-binding motifs in proteins where the ligands responsible for binding have been identified. Zn sites are either of the tetradentate or tridentate type (Vallee and Auld, 1990). Tetradentate sites are found in small Zn-binding domains (zinc fingers) in which the cation is strongly held by four ligands composed of Cys and/or His (Schabe and Klug, 1994). These motifs have affinities of the order of 10M or more (Zeng et al., 1991). The tridentate type of site, found in different extracellular (Vallee and Auld, 1990) and intracellular enzymes (Perlman and Rosner, 1994), bind Zn with an affinity constant of 2 10^6M (Francis et al., 1994). In these enzymes Zn is held by three ligands: two His residues in a typical H-E-x(2)-H or H-x(3)-H sequence implanted on a alpha-helical segment and a third coordinating Glu residue, located at a variable distance, up- or downstream, of the His motif (Vallee and Auld, 1990). S100A8, S100A9, and calgranulin C display such a motif and bind Zn with an affinity of at least 10^8M. The single Zn site is clearly distinct from the two Ca-binding sites. However, other S100 proteins do not possess one of the motifs frequently encountered in proteins where Zn binding has been proven to be functionally important. Strong binding of 8 Zn ions per dimer was also reported for S100B (Baudier et al., 1986). S100A1 (Leung et al., 1987) binds Zn with a rather low affinity. S100A6 (calcyclin) binds Zn with a [Zn](0.5) of about 2 mM (Filipek et al., 1990; Pedrocchi et al., 1994b). But S100A3 is the only S100 protein with 10 Cys residues, most of which are clustered at the opposite site of the two Ca-binding loops. This abundance of sulfur atoms and the fact that the fully reduced and alkylated protein does not bind Zn at all anymore^3 suggest that the Zn ions are bound in thiolate clusters of the Kagi and Kojima type (reviewed in Vallee and Auld(1990)). Direct binding of Zn to the Cys residues also would explain the strong reduction of the thiol reactivity in the presence of Zn, but not of Ca. The metallothioneins bind 7 Zn per mol (8 for the S100A3 dimer) to 20 cysteinyl residues in clusters of the type Zn(3)S(9) and Zn(4)S. The Zn-thiolate cluster has recently also been observed in DNA-binding proteins (Pan and Coleman(1990). However, since Co-binding does not induce the characteristic absorption bands at 700 nm as it does in metallothioneins, and since half of the Cys residues in S100A3 are not in the free thiol form, it is tempting to postulate that a new type of cluster is present in the latter protein. Structural work is in progress to provide a more detailed description of this novel Zn-binding motif.


FOOTNOTES

*
This work was supported by the Wilhelm Sander-Stiftung (FRG), Paul-Martini-Stiftung der Medizinisch-Pharmazeutischen Studiengesellschaft e.v., and the Swiss National Science Foundation Grants 31-37575.93 and 31-40237.94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pediatrics, Division of Clinical Chemistry, University of Zurich, Steinwiesstr. 75, CH-8032 Zurich, Switzerland. Tel.: 41-1-266-7541; Fax: 41-1-266-7169; HeizmannC{at}wawona.vmsmail.ethz.ch.

(^1)
The abbreviations used are: CaBP, calcium-binding protein of the EF-hand family; DTNB, 5,5`-dithiobis(2-nitrobenzoic acid); TNS, 2-p-toluidinylnaphthalene-6-sulfonate; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MBP, maltose-binding protein; ESI, electrospray ionization; MS, mass spectrometry; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography.

(^2)
New nomenclature of S100 proteins: S100 calcium-binding protein A3 (S100E) (see Schäfer et al. (1995)).

(^3)
J. A. Cox, unpublished observations.


ACKNOWLEDGEMENTS

We thank I. Durussel for technical assistance, S. Holm for carrying out the amino acid analysis, Drs. P. Hunziker and D. Bürgisser for the ESI-MS analyses, Dr. B. Schwendiman for help with data analysis, and M. Killen and T. Petrova for correcting the manuscript.


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