The role of cysteine residues in S100B dimerization and regulation of target protein activity1

Biochimica et Biophysica Acta 1343 Ž1997. 117–129

The role of cysteine residues in S100B dimerization and regulation of target protein activity 1 Aimee Landar a,) , Tonya L. Hall a , Emily H. Cornwall a , John J. Correia b, Alexander C. Drohat c , David J. Weber c , Danna B. Zimmer a a

Department of Pharmacology, UniÕersity of South Alabama College of Medicine, Mobile, AL 36688, USA Department of Biochemistry, UniÕersity of Mississippi Medical Center, 2500 North State, Jackson, MS 39216, USA Department of Biochemistry and Molecular Biology, UniÕersity of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA b

c

Received 11 June 1997; accepted 16 July 1997

Abstract Previous studies have demonstrated that the two cysteine residues in the calcium-binding protein S100B are required for its extracellular functions. In the present study, a recombinant S100B protein and mutant S100Bs containing one or no cysteine residueŽs. have been used to determine the contribution of cysteine residues to S100B dimerization and interaction with the intracellular target proteins aldolase, phosphoglucomutase, and the microtubule associated tau protein. Mutation of C68 to a valine or C84 to a serine, C68 to valine and C84 to serine, or C68 to valine and C84 to alanine did not significantly alter S100B activation of aldolase. However, mutation of C84 to serine resulted in calcium-independent S100B activation of phosphoglucomutase and a loss of S100B inhibition of tau phosphorylation by Ca2qrcalmodulin-dependent protein kinase II. The altered functionality of the C84S mutant with phosphoglucomutase and tau was not due to altered physical properties or dimerization state. All of the mutants exhibited heat stability and calcium dependent conformational changes which were identical to recombinant S100B. In addition, S100B proteins containing two, one or no cysteine residues behaved as dimers in size exclusion chromatography experiments in the presence or absence of calcium as well as in the presence or absence of reducing agent. Dynamic light scattering and analytical ultracentrifugation experiments confirmed that dimerization was not affected by calcium or reducing agent. Altogether these results demonstrate that S100B dimerization is not calcium- or sulfhydryl-dependent. In summary, cysteine residues are not necessary for the noncovalent dimerization of S100B, but are important in certain S100B target protein-interactions. q 1997 Elsevier Science B.V. Keywords: S100B; Calcium-binding protein; Cysteine; Dimerization; Target protein activity

Abbreviations: TNS, 2-p-toluidinylnaphthalene-6-sulfonate; IPTG, isopropyl thio-b-D-galactoside; BME, b-mercaptoethanol; EDTA, ethylenediamine tetraacetic acid; EGTA, wethylenebis-Žoxyethylenenitrilo.x-tetraacetic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol ) Corresponding author. Fax: q1 Ž334. 460-6798; E-mail: [email protected] 1 These studies were supported by grants from the National Institutes of Health ŽNS 30660 to DBZ; AG10208 to LVE; and GM 52071 to DJW., National Science Foundation ŽBIO-920038 to DBZ; and BIR-9216150 to JJC., and SRIS and DRIF funding from the State of Maryland. 0167-4838r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 1 2 6 - X

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1. Introduction The S100 proteins are a family of acidic, calciumbinding proteins with approximately 15 members w1x. S100A1 and S100B were the first members of this family to be identified and they share approximately 50% homology in amino acid sequence. These proteins exist as homologous ŽA1–A1, B–B. and heterologous dimers ŽA1–B. w2x. Each S100A1 and S100B subunit contains two EF-hand calcium-binding domains. Binding of calcium results in a conformational change which exposes a hydrophobic patch that can be monitored by the fluorescent probe TNS. This probe associates weakly with hydrophobic residues of proteins such as calmodulin w3,4x, S100A1 w5,6x and S100B w5,7x in the absence of calcium. In the presence of calcium, this probe interacts with the residues of the exposed hydrophobic patch resulting in a stronger interaction and increased fluorescence. Chromatography on hydrophobic resins such as phenyl-Sepharose and native gel electrophoresis can also be used to monitor this calcium-dependent conformational change. Calcium-modulated proteins like S100 have no known enzymatic activity and are postulated to function by interacting with other proteins, termed target proteins, via this hydrophobic patch. However, reports that S100 proteins modulate some target proteins in the absence of calcium suggest that siteŽs. other than the hydrophobic patch participate in target protein interaction Ž see w1x.. S100A1 and S100B have been implicated in numerous cellular processes including cell–cell communication, cell structure, cell growth, energy metabolism, contraction, and intracellular signal transduction w1x. This diversity of function is accomplished by their interaction with a variety of target proteins including fructose-1,6-bisphosphate aldolase w8,1x, phosphoglucomutase w9x, and the microtubuleassociated tau protein w10x. S100 activation of the aldolase A isozyme is not calcium-dependent, while S100 activation of the aldolase C isozyme w8x and modulation of phosphoglucomutase w9x are calciumdependent. S100 interaction with the microtubule associated tau protein results in the inhibition of tau protein phosphorylation by Ca2qrcalmodulin-dependent kinase II w10x. Tau binds to S100B-Sepharose columns in the presence of calcium and is eluted in the presence of EGTA, indicating that the S100B-tau

interaction is calcium-dependent w11x. The large number of diverse target proteins, isozyme specificity of S100 effects, and differences in calcium dependency of S100-target protein interactions suggest multiple mechanisms of target protein interaction. The tertiary structure of S100B has been reported recently w12,13x. While these two studies were performed using S100B from different species, rat w12x and bovine w13x, both show similar structures for the noncovalent, calcium-free S100B dimer. The two regions of S100B with the least amount of sequence homology to other S100-family proteins are the ‘‘hinge region’’ Ž loop II, residues 41–49. and the C-terminal loop Žloop IV, residues 83–91. w12x. Both of these loops are proximal to a large cleft in the protein w12x, and residues in these loops are hypothesized to be responsible for the binding of S100B to target proteins w12,14x. Interestingly, it has been shown that cysteine-84, which is located in a cleft in the C-terminal loop Ž loop IV. , is exposed to solvent upon the addition of Ca2q w5x. Cysteine 68 is also nearby this cleft in S100B, and it lies in a small b-strand which is part of a b-sheet that brings the two EF-hand calcium binding domains together w12x. Either C68 or C84 forms a disulfide linkage with the microtubule-associated tau protein w10x. However, there is no direct evidence demonstrating whether C84 andror C68 Žor disulfide bonds between these residues. are important for the activation of intracellular enzymes by S100B. To directly determine if disulfide bonds or cysteine residues participate in the functioning of the S100B molecule, we have examined the properties of recombinant S100B proteins containing single and double cysteine substitutions. In order to minimize other effects, conservative amino acid substitutions have been made and the cysteine residues have been replaced with valine, serine or alanine residues Ž Fig. 1. . The effects of the cysteine mutations on the functioning of S100B have been analyzed using the S100B target proteins fructose-1,6-bisphosphate aldolase A and C, phosphoglucomutase, and the microtubule associated tau protein. These studies demonstrate that mutation of C84 to serine results in calcium-independent activation of phosphoglucomutase, as well as loss of S100B inhibition of tau phosphorylation. Furthermore, altered functionality of the cysteine mutants could not be attributed to altered physical prop-

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Fig. 1. Linear diagram of recombinant and mutant S100B proteins. The cysteine residues present in recombinant S100B ŽVUSB-1. are located at positions 68 and 84. Mutant proteins are shown below VUSB-1 with the amino acid changes indicated using the single letter code. Semicircles represent EF-Hand calcium-binding motifs.

erties, including heat stability, calcium-dependent conformational changes, or dimerization. Altogether, our results suggest that cysteine residues are not required for noncovalent S100B dimerization, but are important in certain S100B-target protein interactions.

2. Materials and methods 2.1. Purification of proteins Transformants containing plasmids encoding recombinant bovine S100B protein ŽVUSB-1. and mutants containing single cysteine changes ŽC84S and C68V. and double cysteine changes ŽC68V84S and C68V84A. were the generous gift of Dr. Linda Van Eldik ŽNorthwestern University School of Medicine, Chicago, IL. w15,16x. A single colony from a LB agar ŽGibcorBRL, Gaithersberg, MD. plate containing 75 mgrml ampicillin Ž Sigma, St. Louis, MO. was used to inoculate 500 ml of LB media Ž Gibco. containing 75 mgrml ampicillin and 0.5 mM IPTG ŽFisher Scientific, Fair Lawn, NJ.. After incubation at

378C with shaking for 16 h, the cells were harvested by centrifugation at 17 700 = g for 15 min and the cell pellet frozen at y208C. Pellets were resuspended in lysis buffer Ž5 mM EDTA, 50 mM Tris, pH 7.5. and treated with lysozyme Ž 6 mgrml.. Cellular debris was removed by centrifugation at 15 000 = g for 15 min and the supernatant boiled for 10 min. After centrifugation at 15 000 = g for 15 min, the supernatant was adjusted to 10 mM CaCl 2 and applied to a 5 ml phenyl-Sepharose ŽPharmacia, Piscataway, NJ. column. The column was washed with equilibration buffer Ž 5 mM CaCl 2 , 1 mM BME, 50 mM Tris, pH 7.5., followed by equilibration buffer containing 500 mM NaCl. Bound proteins were eluted with 5 mM EDTA, 1 mM BME, 50 mM Tris, pH 7.5, dialyzed extensively against 50 mM Tris, pH 7.5, and stored at y208C. Purity was assessed by SDS–polyacrylamide gel electrophoresis. Recombinant rat S100B was prepared from an overexpression plasmid in Escherichia coli cells ŽHMS174DE3. as described previously w17x. Aldolase A and phosphoglucomutase were purchased from Boehringer Mannheim Ž Indianapolis, IN.. Full-length tau protein was recombinantly expressed in E. coli n123c clones Ža generous gift of Dr. Gloria Lee, Harvard Medical School, Boston,

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MA., and protein was purified as previously described w18x. 2.2. Non-denaturing gel electrophoresis Samples in 7.5% glycerol, 0.03% bromophenol blue, 0.05% DTT, 0.04 M Tris, pH 6.8, were electrophoresed in 15% polyacrylamide gels containing no SDS and either 10 mM CaCl 2 or 10 mM EDTA w19x. After electrophoresis at 100 V in 0.192 M glycine, 0.025 M Tris-base containing either 10 mM CaCl 2 or 10 mM EDTA, gels were stained with Coomassie Brilliant Blue. 2.3. Size exclusion chromatography A G75 column Ž4.91 cm2 = 91.5 cm. was equilibrated with several column volumes of buffer Ž10 mM Tris-HCl, 50 mM NaCl, 2 mM BME, pH 7.40. with and without 2.5 mM CaCl 2 . All of the buffers in the calcium free experiments were previously treated with chelex-100 to remove divalent metal ions. Next, the column was calibrated for molecular weight determinations using low molecular weight protein standards ŽPharmacia.. The standard proteins included ribonuclease A ŽMW 13.7 kDa. , chymotrypsinogen A ŽMW 25.0 kDa. , ovalbumin Ž MW 43.0 kDa. , and bovine serum albumin Ž67.0 kDa. . Disulfide-linked recombinant rat S100B dimer was prepared as previously described w15x and used as protein standard. In each case, 1 ml of the protein standard Ž10 mgrml. was loaded onto the column, and 3 ml fractions were collected and monitored with A 280 readings. Dextran blue was applied to the G75 column to determine its void volume. The K av values Ž K av s w Ve y Vo xrw Vt y Vo x. were determined for each protein and plotted versus the log of the molecular weight of the standard where Ve is the elution volume at the peak apex, Vo is the void volume, and Vt is the total column volume. Similarly, 1 ml samples of reduced S100B Ž1–9 mM; 10–90 mgrml. were applied to the same column and a K av value was determined in each case. In experiments with calcium present, S100B Ž1 mM; 10 mgrml. was incubated with 2.5 mM Ca2q for 1 h prior to loading the protein onto a column containing buffer and 2.5 mM Ca2q. The molecular weight of S100B was determined by comparing its

K av value to those found for the standard proteins as previously described w20x. FPLC analysis using a prepacked Superose 12 column ŽPharmacia. was used to determine the dimerization of recombinant bovine S100B and all mutant S100B proteins. Proteins were extensively dialyzed against distilled water and filtered through a 22 mm syringe filter. The column was equilibrated with several volumes of 10 mM Tris, 50 mM NaCl, 2 mM BME, pH 7.4 with 2.5 mM CaCl 2 or 1 mM EDTA Ž calcium chelating agent. , and calibrated using the same low molecular weight standards described above. A 0.4 mlrmin flow rate was used to allow sufficient separation. The void volume was estimated as recommended by the manufacturer to be 35% of the column bed volume, and K av values and molecular weights were calculated as described above. 2.4. Dynamic light scattering Dynamic light scattering is also a standard technique used to determine the apparent molecular weight of globular proteins w21x. The diffusion coefficient Ž D T . of recombinant rat S100B was determined by measuring light scattering as a function of S100B concentration using a dp-801 Biotage spectrometer ŽBiotage, Charlottesville, VA.. Assuming Brownian motion, the hydrodynamic radius of S100B could be calculated using the Stokes–Einstein equation: R H s k bTr6ph D T

Ž1.

where k b is the Boltzmann’s constant, T is absolute temperature, and where h is solvent viscosity. The apparent molecular weight of S100B could then be estimated based on a standard curve of R H versus molecular weight of twenty standard proteins w22x. Measurements were completed at each of four S100B concentrations: 20, 13, 6.7, and 3.3 mgrml Ž2, 1.3, 0.67, and 0.33 mM. . All S100B solutions contained 25 mM NaCl, 5.0 mM BME, 4.0 mM Tris-HCl, pH 7.5, and they were filtered through a 20 nm syringe filter to remove any dust particles. Data analysis to give accurate molecular weight values were performed using two methods as previously described w23x. The first method uses only the lowest S100B concentration points as accurate measurements of the molecular weight since these points have a negligible amount of polydispersity. Alternatively, all of the

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apparent molecular weight values at each S100B concentration were used, and a plot of apparent molecular weight versus S100B concentration was used to extrapolate to the molecular weight of S100B at an infinite dilution to minimize effects of polydispersity w23x. 2.5. Analytical ultracentrifugation All experiments were conducted on a Beckman Optima XLA analytical ultracentrifuge equipped with absorbance optics and an An60Ti rotor. Temperature was calibrated using 0.1 M CoCl 2 in 92.5% ethanol as described w24x. All experiments were done at a setting of 258C which was found to correspond to 24.68C. Sedimentation equilibrium experiments were performed at 48 000 rpm in charcoal filled epon six channel centerpieces. Loading concentrations were 0.05–0.2 OD 280 corresponding to approximately 35– 150 mM Ž0.35–1.5 mgrml.. Equilibrium data were collected at an appropriate wavelength Ž 242 nm and 280 nm. and at a spacing of 0.001 cm with 16 averages in a step scan mode. Equilibrium was checked by comparing scans at various times up to 24 h. Data sets were edited with REEDIT Ž Jeff Lary, National Analytical Ultracentrifuge Center, Storrs, CT. to extract the three channels of data, and fit individually and jointly with NONLIN w25x Žsee Section 3. . NONLIN fits to an effective reduced molecular weight, s s M Ž1 y yr . v 2rRT, where M is the molecular weight, y is the partial specific volume, r is the solvent density, v s Ž 2 p rpmr60. , R is the gas constant and T is the temperature in Kelvin w26x. Fits to a single species give a Z-average s and thus a Z-average molecular weight, Mz w27x. The sequence derived molecular weights are 10 536 for bovine recombinant S100B, 10 500 for C68V84A, and 10 516 for C68V84S. The 95% confidence intervals on parameters estimated by NONLIN were determined by a global search, and are presented as error limits corresponding to " 2 standard deviations w25x. The molecular weights reported in Table 1 are whole cell averages derived from single species fits. Single species fits are the best fits for these data Ždata not shown.. A reversible mechanism of monomer-dimer or nonideal monomer-dimer gives worse fits suggesting a nonequilibrium mixture of S100B monomers and dimers. All experiments were done in 50 mM

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Table 1 Summary of sedimentation equilibrium data with S100B, C68V84A and C68V84S a,b Ca2q DTT MZ

² y, q : c

rms d % Dimer e y3 Ž=10 . wgt fraction

S100B y y y y q y y q q q

18202 18322 18936 f 18035 g 18289 h

²17599, 18820: ²17578, 19092: ²18199, 19699: ²17363, 18729: ²17632, 18961:

4.09 5.79 4.59 5.68 5.21

57.2 58.6 66.3 55.2 58.2

C68V84A y y q y

16778 ²15954, 17616: 6.16 17230 f ²16542, 17921: 5.21

42.6 47.2

C68V84S y y q y

16318 ²15732, 16915: 4.83 17450 f ²16791, 18128: 4.75

38.1 49.2

a

Global fits of three channels of data collected at 280 nm and jointly with Nonlin as described in Section 2. Nearly identical results were obtained with data collected at 242 nm. All experiments done at 24.68C in 50 mM Tris, pH 7.5 and the indicated additives. b Loading concentrations were approximately 0.35 to 1.5 mgrml protein Žsee Section 2.. c Corresponds to a 95% confidence interval or "2 standard deviations. d Root mean square deviation of the global fit in OD units. e Weight fraction of dimer as derived from the Mz from the global fit. f Done in 2 mM CaCl 2 plus 1 mM EDTA, 50 mM Tris. g Done in 0.1 mM DTT, 50 mM Tris. h Done in 0.1 mM DTT, 2 mM CaCl 2 , 1 mM EDTA, 50 mM Tris.

Tris, pH 7.5 " 1 mM EDTA, 2 mM CaCl 2 " 0.1 mM DTT. The density of the buffer was determined in a Mettler-Parr DMA 02D precision density meter to be 0.99870 gmrml at 24.68C. The partial specific volume of each construct was calculated by the method of Cohen and Edsall to be 0.731 for S100B and 0.734 for the double mutants C68V84A and C68V84S w28x. The binding of Ca2q may also alter y and neglecting this effect will cause an underestimate of molecular weight, although the magnitude is obviously dependent upon the extent of binding w28x. Because the temperature was calibrated and the buffer density was measured, the single largest source of error in these molecular weight measurements is the y estimation. It is generally accepted that the Cohen and Edsall values give the most reliable molecular weights w29x.

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2.6. Target protein assays

3. Results

Recombinant bovine S100B and mutant S100B stimulation of aldolase activity was assayed as previously described w8x using a commercially available colorimetric assay kit a752 from Sigma ŽSt. Louis, MO. which is based on the procedure of Sibley and Lehninger w30x. Recombinant bovine S100B and mutant S100B stimulation of phosphoglucomutase activity was assayed as described w9x, using the standard phosphoglucomutase assay described by Boehringer Mannheim. In both assays, recombinant and mutant S100B concentrations ranged from 10y8 to 10y5 M Ž0.1–100 mgrml. . This concentration range encompasses the previously reported S100B dose response curves w8,9x. Data from the aldolase and phosphoglucomutase experiments are expressed as the fold increase over basal activity. The tau phosphorylation assay was based on the procedure of Baudier and Cole w10x. The final reaction volume was 30 ml containing 13–26 mM Ž0.13–0.26 mgrml. recombinant bovine S100B or mutant S100B protein, 0.25 mM tau, 1 mM CaCl 2 , 20 mM Tris, pH 7.5, 10 mM DTT, 4 mM MgCl 2 , 40 mM w g-32 Px-ATP Ž10 Cirmmol., 2.4 mM calmodulin, and 0.33 ng recombinant Ca2qrcalmodulin-dependent kinase II Ž New England Biolabs, Beverly, MA.. S100B proteins were preincubated 30 min on ice with tau, CaCl 2 , and buffer. Ca2qrcalmodulin-dependent kinase II was preincubated 5 min on ice with DTT, MgCl 2 , CaCl 2 , buffer, and w g-32 Px-ATP. The S100B and kinase containing solutions were mixed and incubated at 378C for 10 min. The reaction was terminated by the addition of 6 ml sample buffer Ž200 mM Tris, pH 6.8, 5% SDS, 38% glycerol, 250 mM DTT, 0.03% bromophenol blue. followed by boiling for 5 min. Samples were size-fractionated on 10% SDS–polyacrylamide gels. The gels were dried and scanned on a Bio Rad GS 250 PhosphoImager ŽHercules, CA.. The percent tau phosphorylation was calculated as the Ž 32 P incorporated in the presence of S100r32 P incorporated in the absence of S100. = 100, and expressed as the mean " the standard error of the mean. Data from all target protein experiments were analyzed using ANOVA tests Ž Graph Pad Instat, Graph Pad Software, San Diego, CA. appropriate for the number of samples and the distribution of the data Ž see Figures for p values..

3.1. Physical properties of mutant S100Bs Previous studies by Van Eldik and coworkers w15,16x did not determine if mutation of one or both cysteine residues altered the ability of S100B to undergo a change in environment in response to calcium binding. To address this question, mutant S100Bs were subjected to phenyl-Sepharose chromatography, native gel electrophoresis, and TNS-binding assays. Recombinant bovine S100B Ž VUSB-1. and cysteine mutant cell pellet lysates contained a distinct band on SDS–polyacrylamide gels with a subunit molecular weight of 10 000 Da. These proteins were heat-stable and bound phenyl-Sepharose in a calcium-dependent manner Ždata not shown. . In addition to exhibiting calcium-dependent interaction with phenyl-Sepharose, mutant S100Bs containing one or no cysteine residues exhibited calcium-dependent increases in TNS fluorescence Ž data not shown.. The TNS titration curves for all mutants were broad and indistinguishable from those previously reported for S100B w31,7x. The C68V, C84S, C68V84A, and C68V84S mutants also exhibited migrations in nondenaturing gels containing EDTA Ž Fig. 2Ž A.. or calcium ŽFig. 2ŽB.. which were indistinguishable from

Fig. 2. Native gel electrophoresis of recombinant and mutant S100Bs. Recombinant S100B ŽVUSB-1. Žlanes 1., C68V Žlanes 2., C84S Žlanes 3., C68V84S Žlanes 4., and C68V84A Žlanes 5. were electrophoresed on non-denaturing 15% Žwrv. polyacrylamide gels in the presence of 10 mM EDTA ŽPanel A. or 10 mM CaCl 2 ŽPanel B..

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recombinant S100B Ž VUSB-1. Ž Fig. 2. and native S100B Ždata not shown. . Thus, mutation of the C68 andror C84 cysteine residues did not alter two properties of S100B: heat stability and calcium-induced conformational change. 3.2. Molecular weight determination In order to monitor the dimerization of S100B, dynamic light scattering and gel permeation experiments were performed with recombinant S100B. Dynamic light scattering studies demonstrated that recombinant rat S100B has an average molecular weight of 21 500 " 1200 Da under reducing conditions at concentrations ranging from 3.3–20 mgrml Ž0.33– 2 mM. Ž Fig. 3ŽB.. . Gel filtration chromatography experiments were performed using recombinant bovine S100B and mutant S100B proteins in the presence of reducing agent and in the presence of calcium or calcium chelating agent. Under either condition, recombinant bovine S100B eluted as a single peak from the column at a molecular weight of approximately 23 000 Da Ždata not shown. , confirming that reduced S100B dimerizes in a calcium-independent

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manner. Similarly, recombinant rat S100B protein was also found to elute from gel filtration columns as a single peak with a molecular weight of a dimer Ž25 100 " 4100 Da. when compared to the elution profile of proteins of known molecular weight Ž Fig. 3ŽA. and ŽB... Furthermore, no difference in the elution profile of reduced S100B and oxidized, disulfide-linked S100B dimer could be detected indicating that reduced subunits of S100B also exist as a dimer. Under denaturing conditions Ž 6 M urea or 6 M guanidinium hydrochloride., the S100B subunit eluted at a volume consistent with its calculated molecular weight of 10 500 Da. Interestingly, neither protein changes its elution profile in gel permeation chromatography experiments when Ca2q is added. This suggests that no aggregation or disaggregation occurs upon binding Ca2q ions at low S100B concentrations. Each mutant S100B protein eluted from the column with a molecular weight of approximately 23 000 Da under reducing conditions in the presence of calcium or calcium chelating agent Ž data not shown.. These results confirm that dimerization is not due to Ca2q ion induced changes in hydrophobicity or disulfide bonds.

Fig. 3. Size-exclusion chromatography of recombinant S100B. ŽA. Elution profile of S100B from a G-75 gel permeation chromatography column Žsee Section 2 for conditions.. Arrows indicate the elution volumes for the 13.7, 25.0, 43.0, and 67.0 kDa molecular weight standards, and the asterisk indicates the elution volume of the disulfide-linked dimer of S100B as an additional standard. ŽB. The K av value is plotted versus the log of the molecular weight of the standard proteins. K av s w Ve y Vo xrw Vt y Vo x where Ve is the elution volume that contains the maximum amount of the protein Žsee ŽA.., Vo is the void volume of the column, and Vt is the total column volume. The dashed line indicates the K av value obtained and the extrapolated log of the molecular weight for S100B.

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Dimerization of the S100B molecule was also determined by analytical ultracentrifugation. Analytical ultracentrifugation studies at protein loading concentrations ranging from 0.35–1.5 mgrml Ž35– 150 mM. demonstrated that recombinant S100B had a Z-average molecular weight of 18 200–18 300 Da, as indicated by replicate experiments Ž Table 1. , suggesting that it exists as a dimer in the absence of calcium. The molecular weight was unchanged in the presence of 2 mM Ca2q, 1 mm EDTA Ž18 900 Da. or 0.1 mM DTT Ž18 000–18 300 Da.. The average value for all experimental data collected was 18 000 " 500 Da Ž242 nm data not shown. . A similar molecular weight was observed when S100B was prepared in the absence of reducing agent and analyzed in the presence or absence of 0.1 mM DTT Ždata not shown.. While there was a small increase in molecular weight in many of the samples due to the addition of Ca2q, it is within the experimental uncertainty of the measurements. Interestingly, 1 mM Zn2q caused S100B to precipitate Ždata not shown. . These results demonstrate that calcium andror DTT do not change S100B dimerization. The molecular weights of S100B determined by this method are less than would be expected for complete dimerization of S100B. The fact that S100B behaves exclusively as a dimer by gel

permeation and dynamic light scattering Fig. 4 indicates that the data obtained by analytical ultracentrifugation may not accurately reflect the dimerization state of S100B. S100B mutants lacking cysteine residues were also analyzed to rule out the possibility of reducing agent insensitive disulfide bridges. When compared to S100B, C68V84A and C68V84S exhibited slight changes in Z-average molecular weights in the presence of calcium Žsee Table 1. . However, these changes fall within the experimental uncertainty of the method. These data indicate that the S100B mutants lacking cysteine residues exist in solution as dimers. Altogether, these biophysical techniques demonstrate that S100B exists as a dimer over a wide concentration range Ž0.035–2 mM. , and that the cysteine residues have little or no role in S100B dimerization. While S100B by itself exists as a dimer, it is possible that target protein interaction may influence the dimerization state of S100B. 3.3. Mutant S100B protein modulation of target proteins Consistent with the view that S100 proteins modulate rather than instigate calcium signal transduction pathways, these proteins often interact with target

Fig. 4. Dynamic light scattering of recombinant S100B. A plot of apparent molecular weight versus S100B concentration is plotted and extrapolated to a molecular weight value at infinite dilution in order to correct for polydispersity effects in the light scattering measurements. See Section 2 for conditions.

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proteins stoichiometrically and modulate their activity by 0.25–3 fold w1x. In order to determine if cysteine residues are required for S100B modulation of intracellular target proteins, the ability of mutant S100B molecules containing single cysteine residues to modulate target protein activity was measured. While they do not exactly mimic the in vivo environment, in vitro target protein assays are useful in identifying regions of the S100B molecule which participate in target protein interaction. As shown in Fig. 5, VUSB-1 exhibited a 1.59 " 0.05 fold stimulation of phosphoglucomutase activity which was calcium-dependent. These data are consistent with previously published levels of S100B stimulation of phosphoglucomutase activity w9x. C68V and both double cysteine mutants exhibited stimulations which were also calcium-dependent and not significantly different from that observed for similar concentrations of VUSB-1. However, the C84S mutant stimulated phosphoglucomutase activity approximately 1.5 fold in the presence and absence of calcium. The dose–response curves for VUSB-1 and the mutant S100B proteins were indistinguishable Ž data not shown. . Thus, the C84S mutant retains the ability to stimulate phosphoglucomutase activity, but it loses calcium dependency. These results suggest that cysteine

Fig. 5. Stimulation of phosphoglucomutase activity by mutant S100B proteins. The ability of recombinant S100B ŽVUSB-1., C68V, C84S, C68V84S, or C68V84A to stimulate phosphoglucomutase activity was analyzed in the presence of 5 mM calcium Žopen bars. or 5 mM EGTA Žgray bars.. Phosphoglucomutase activity obtained in the absence of S100 was normalized to 1.0, and the activity obtained in the presence of S100 expressed as the mean fold stimulation " the standard error of the mean. Values represent the mean of at least 7 determinations. ) ) indicate one tailed p values F 0.001.

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Fig. 6. Inhibition of tau protein phosphorylation by mutant S100B proteins. The ability of recombinant bovine S100B ŽVUSB-1., C68V, C84S, C68V84S, or C68V84A to inhibit tau protein phosphorylation was determined in the presence of 1 mM calcium. Tau protein phosphorylation obtained in the absence of S100 Žcontrol. was normalized to 100%, and inhibition obtained in the presence of S100 expressed as the mean percent tau phosphorylation " the standard error of the mean. Values represent the mean of at least 4 determinations. The only mutant protein which exhibited a significant change in percent tau phosphorylation when compared with recombinant bovine S100B was C84S. ) ) indicate a one tailed p value F 0.001.

residues are important in determining the calcium dependency of S100B stimulation of phosphoglucomutase. In order to determine the importance of S100B cysteine residues with non-enzyme target proteins, the inhibition of tau phosphorylation by Ca2qrcalmodulin-dependent kinase II was analyzed. VUSB-1 inhibited tau phosphorylation to 65.1 " 7.4% of control, which is less than that reported by Baudier and Cole w10x. Nonetheless, the level of S100 inhibition of tau phosphorylation in our experiments was sufficient to detect differences in S100B mutant proteins. At similar concentrations, inhibition of tau phosphorylation by C68V, C68V84S, and C68V84A Ž78.7 " 9.1%, 78.0 " 7.1%, and 76.7 " 5.7% of control, respectively. were not significantly different from VUSB-1 ŽFig. 6.. However, equivalent concentrations of the C84S mutant did not inhibit and may have slightly stimulated tau protein phosphorylation Ž 108.5 " 7.0% . . These results demonstrate that cysteine residues are important in S100B modulation of tau protein phosphorylation. The calcium dependency of this effect could not be examined since calcium is required for the proper

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functioning of the Ca2qrcalmodulin-dependent kinase II in the assay. In order to determine the universality of cysteine residues in S100B-target protein interaction, the abilities of mutant S100Bs to stimulate various aldolase isozymes were measured. The aldolase A isoform is stimulated by S100B in a calcium-independent man-

ner, while the aldolase C isoform is only stimulated by S100B in the presence of calcium w8x. As shown in Fig. 7ŽA. , VUSB-1 as well as mutant S100Bs containing one or no cysteine residues stimulated aldolase A activity in the presence of 5 mM Ca2q approximately 2.5 fold. While VUSB-1 and mutant proteins exhibited lower stimulation in the presence of EDTA, these differences were not statistically significant Ž p ) 0.05.. As shown in Fig. 7Ž B. , calcium-dependent activation of aldolase C by mutant S100Bs containing two, one, or no cysteine residues was not significantly different from VUSB-1. Furthermore, there were no detectable differences between the dose-response curves for the single and double cysteine mutants when compared to S100B Ždata not shown.. The levels of stimulation observed in this study are in agreement with previously published fold stimulations of aldolase A and C by S100B w8x. These results demonstrate that cysteine residues are not obligatory for calcium-independent activation of aldolase A or the calcium-dependent activation of aldolase C by S100B. Thus, the functional effects of mutating the cysteine residues in S100B are target protein-dependent.

4. Discussion

Fig. 7. Stimulation of aldolase A and C activities by mutant S100B proteins. The ability of recombinant bovine S100B ŽVUSB-1., C68V, C84S, C68V84S, or C68V84A to stimulate aldolase A activity ŽPanel A., and aldolase C activity ŽPanel B. was analyzed in the presence of 5 mM calcium Žopen bars. or 5 mM EDTA Žgray bars.. Aldolase activity obtained in the absence of S100 was normalized to 1.0, and the activity obtained in the presence of S100 expressed as the mean fold stimulation " the standard error of the mean. Values represent the mean of at least 5 ŽPanel A. or 2–4 ŽPanel B. determinations. ) and ) ) indicate one tailed p values - 0.05 and - 0.001, respectively.

This is the first study which directly investigates the contribution of cysteine residues 68 and 84 to S100B interaction with intracellular target proteins. Mutation of C68 to valine did not alter S100B modulation of aldolase A, aldolase C, phosphoglucomutase or tau protein suggesting that it is not involved in target protein modulation. In contrast, mutation of C84 to a serine resulted in a loss of tau protein modulation and altered calcium-dependency of phosphoglucomutase stimulation. However, mutation of C84 did not alter stimulation of aldolase A or C. Altogether, these data demonstrate that cysteine 84 participates in the modulation of some, but not all, S100B target proteins.The variable effects of the mutants on different target proteins is consistent with previous studies on calmodulin which suggest that calmodulin does not bind all of its target proteins in the same way w32,33x. Before a general mechanism for S100-target protein interaction can be proposed, it

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will be necessary to examine S100 interaction with a variety of target proteins. Our observations that all of the mutant proteins exhibit calcium-dependent mobility shifts in non-denaturing gels, binding to the fluorophore TNS, and interaction with phenyl-Sepharose demonstrates that disulfide bonds andror cysteine residues are not involved in calcium-dependent exposure of a hydrophobic patch. If the hydrophobic patch which is exposed in the presence of calcium is the site of target protein interaction, then one would expect none of the mutant S100Bs to exhibit altered modulation of target protein activity. However, it is clear that the calcium-dependent hydrophobic patch is not the only site involved in target protein modulation because S100B modulates some target proteins Ž aldolase A. in the absence of calcium. Thus, our observations of altered C84S modulation of phosphoglucomutase without an alteration in calcium-dependent exposure of a hydrophobic patch were not surprising. These results support the idea that the hydrophobic patch exposed upon calcium binding is not the only site involved in S100B target protein interaction. Since C84S activates phosphoglucomutase in the presence of a calcium chelating agent, whereas S100B does not, it is reasonable to conclude that C84S is structurally distinct from wild-type apo-S100B. Likewise, since C84S loses tau phosphorylation inhibition in the presence of calcium, it is also structurally distinct from wild-type calcium-bound S100B. Interestingly, simultaneous mutation of C68 and C84 residues results in both inhibition of tau phosphorylation and calcium-dependency of phosphoglucomutase activation which are similar to recombinant S100B. A possible explanation for this effect is that a structural perturbation caused by the replacement of C84 to serine is compensated for by mutating C68 to valine. For example, it is possible that mutation of C84 to a hydrophilic serine residue disrupts hydrophobic interactions or forms new hydrogen bonds within the S100B protein or between S100B and its target proteins. Conversely, the simultaneous mutation of C68 to a hydrophobic valine may promote hydrophobic interactions and counterbalance the effects of the serine. The fact that mutation of C84 to serine affects S100B modulation of some, but not all, S100B target proteins is consistent with the view that S100B has more than one site of target protein interaction.

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The recent implication of disulfide bonds in the NEF activity of S100B w16x and the observation of monomeric S100B on SDS–polyacrylamide gels of fractions from gel filtration columns run under reducing conditions w34x have sometimes been interpreted to mean that reduced S100B is a monomer and oxidized S100B is a dimer. The dynamic light scattering and size exclusion chromatography experiments reported in this study confirm the finding that, under reducing conditions, S100B is a dimer w12,13x. In addition, our results confirm that the dimerization motif for S100B is a noncovalent motif which is destroyed in the presence of denaturing agents such as SDS w2,35–38x. We propose the following nomenclature for the S100 protein species: S100B for the monomer, S100BŽ bb . for the noncovalent reduced dimer, and S100BŽ bs – s b . for the oxidized dimer. A fourth species of S100B dimer, termed S100BŽ bb .A , has been observed under reducing conditions in SDS–polyacrylamide gels in a number of laboratories and represents an artifactual dimer produced by incubation in denaturing agents w34x. It is clear that the extracellular NEF activity requires the S100BŽ bs – s b . species which contains disulfide bridges w44,16x and that the S100BŽ bb . species is not sufficient to induce neurite extension. Such a disulfide-bridge dimer might be easily formed in the oxidizing extracellular environment. Though the NMR structures and our data demonstrate a noncovalent dimerization motif for S100B, this does not rule out the possibility of an unrelated disulfide-linked dimerization motif. However, S100B purified under nonreducing conditions behaves almost exclusively as the S100BŽ bb . species both in the presence and absence of reducing agent. These results suggest that only a very small amount of S100BŽ bs – s b . would be produced in the extracellular environment. It is possible that S100BŽ bs – s b . may be secreted directly from glial cells in vivo and, even if it is a minor species, may be capable of producing a biological effect, as many growth factors present at relatively low concentrations are capable of producing biological responses. Isolation and characterization of S100BŽ bs – s b . along with further characterization of the dimerization motifs in S100BŽ bb . will be needed before these issues can be resolved. Due to the possibility of modification during any purification procedure, systems in which the function

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of mutated S100 proteins can be analyzed in intact cells may be the only direct method for answering this question. Nondisulfide-bridge dimerization motifs have been reported for a number of proteins including the herpes simplex virus type 1 immediate early protein ICP4 w39x, the R6K plasmid replication initiator protein p w40x, the rat prostatic acid phosphatase w41x, and interleukin-8 w42x. Furthermore, two three-dimensional solution structures of dimeric rat and bovine apo-S100BŽ bb . were recently determined by NMR spectroscopy under reducing conditions w12,13x. The structure at the dimer interface was shown to adopt a X-type four helical bundle comprising two helices ŽI and IV. from each subunit w12,13x. A large number of conserved residues at this interface indicates that dimerization may be important for the function of S100 proteins in general w43,12x. Currently, the only procedure which will generate pure monomer at micromolar or higher concentrations is treatment with denaturing agents such as SDS, urea, or guanidinium. These agents also destroy target protein activity, making it impossible to determine if the monomer is functional. In summary, the cysteine residuesrdisulfide bridges in S100BŽ bb . are not obligatory for many physical properties of this molecule including heat stability, dimerization, calcium-induced conformational change, and target protein activation. However, cysteine 68 and 84 do contribute to the interaction of S100BŽ bb . with intracellular target proteins. It will be important in future studies to identify the mechanisms involved in S100BŽ bb . heat stability, dimerization and target protein activation. This information will be instrumental in determining how S100B regulates target protein function. This information will ultimately allow for the identification of new pharmacological agents that interfere with S100BŽ bb . function.

Acknowledgements The authors would like to thank Dr. Linda Van Eldik ŽNorthwestern University, Chicago, IL. for the generous gift of the recombinant S100B and mutant expression vectors, Dr. Gloria Lee Ž Harvard Medical School, Boston, MA. for the generous gift of the

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