Structural and ion-binding properties of an S100b protein mixed disulfide: Comparison with the reappraised native S100b protein properties

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 279, No. 1, May 15, pp. 174-182,199O

Structural and Ion-Binding Properties of an Sl OOb Protein Mixed Disulfide: Comparison with the Reappraised Native Sl OOb Protein Properties’ Yves Mely2 and Dominique

Gerard

Universite’ Louis Pasteur, Laboratoire de Biophysique, de Strasbourg, B.P. 24,674Ol Illkirch Ceden, France

Received October 12, 1989, and in revised form January

UA CNRS 491, Faculte’de Pharmacie

23, 1990

SlOOb protein, chemically modified by thioethanol groups (linked via disulfide bonds to two out of four Cys per dimer) was largely similar to reduced native S 1OOb protein in its overall structure and differed only by small modifications extending, however, to the whole protein structure. Studies combining direct Ca2+ binding and associated conformational changes revealed that this chemical modification markedly increased the Cazf-binding affinities (especially in the presence of physiological concentrations of K+ and Mg2+) and introduced a strong positive cooperativity. Different binding models are discussed and it emerges that in both proteins the Ca2’-binding sites are not equivalent and probably interact. Like the reduced protein, chemically modified SlOOb protein binds four Zn2+ ions in two classes of sites (of high and low affinities). Whereas the overall Zn2+ affinity was only slightly decreased, the binding sequence was probably reversed by the introduction of thioethanol groups. Moreover, in the preswere higher and even ence of zinc, the Ca” affinities o 1990Academic PMS, I~C. identical, in both proteins.

SlOOb protein is a low-weight, acidic, noncovalent dimerit (with pp subunit composition) Ca’+-binding protein of the same family as calmodulin, troponin C, parvalbumin, and calbindin 9K (1). Each p subunit (M, 10,500) is characterized by two Ca’+-binding sites of the EF hand type, though the N-terminal binding site is rather a “pseudo” EF hand with two additional amino acids in the Ca’+-binding loop (2). Nevertheless, unlike 1 This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale (Grant 861010) and the Universite Louis Pasteur. ’ Recipient of a fellowship from the Minis&e de la Recherche et des Technologies and to whom correspondence should be addressed. 174

most of the other EF hand-containing Ca2+-binding proteins, SlOOb protein seems to bind Ca2+ only weakly, with affinities incompatible with the intracellular Ca2+ concentrations (3,4). In clear contrast, its Zn2+-binding affinities are rather high and probably physiologically relevant (4). Ion binding to SlOOb protein is accompanied by important conformational changes mainly monitored spectroscopically using either the intrinsic spectroscopic labels (i.e., Tyr 17 and Phe residues) or extrinsic labels coupled to Cys residues (3,5-7). With their high reactivities and their binding capabilities, Cys residues are particularly important protein elements (for review see (8,9)). However, most of the Ca2+binding proteins of the calmodulin family are characterized by only a few or even no Cys residues (10-13). In the case of SlOOb protein, each SlOO/3 subunit includes two Cys residues (l), both located in the C-terminal part of the protein at positions 68 and 84. Not surprisingly, either in the apo-form, or in the presence of K+ or Zn2’ ions, both Cys are embedded in the protein core and can not react (or only slowly) with external thiol reagents (5): such a Cys burial has been commonly reported in many proteins (8). However, in the presence of Ca”, Cys 84 are specifically exposed to the solvent and can react with numerous thiol-specific reagents (5-7, 14, 15). The Ca2+-induced exposure of these reactive Cys seems physiologically relevant since disulfide-bridged complexes between SlOOb protein and protein r (2) of microtubules, have been described (1516). Moreover, such complexes may also be involved (15) in the Ca2’-induced binding of SlOO proteins to membranes (17). In this context, the aim of our work was to engineer a model mixed disulfide derivative and to study the structural and ion-binding changes induced. To this end, a mixed disulfide with thioethanol groups blocking two out of four Cys residues was chosen. This species3 accu’ Mely and Gerard, submitted. 0003.9861/90

53.00

Copyright W 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

PROPERTIES

OF AN SlOOb PROTEIN

MIXED

175

DISULFIDE

mulated readily when SlOOb protein was incubated with 2,2’-dithioethanol in the presence of Cazi.. The blocking thioethanol group has no chromophoric structure and so could not intervene in spectroscopic studies of the intrinsic aromatic residues. Furthermore, this residue is small in size and of medium polarity and so probably could not induce any major steric or charge-repulsive constraints. The ion-binding properties and the associated conformational changes of this chemically modified SlOOb protein were compared with the reinvestigated properties of native reduced protein, which allowed discussion of their ion-binding mechanisms.

ligand, were fitted to the Adair-Klotz equation by a nonlinear square procedure, allowing the determination of the macroscopic ing constants, K,, as described (24),

EXPERIMENTAL

where S, (; = 0 to n) represented the fluorescence intensities associated with the protein species when i ligands are bound. The S, parameters were obtained by fitting Eq. [2] with a nonlinear least square procedure, using the macroscopic binding constants, K,, and the free ion concentration, .r, determined from binding experiments. All calculations and curve-fitting were carried out using SAS computer procedures.

PROCEDURES

Materials Native, reduced SlOOb protein was prepared as described (18). The mixed disulfide derivative” was obtained from reduced SlOOb protein by incubation with 20 mol excess of 2,2’-dithioethanol, in the presence of 1 mM Ca”, and then separated from the reduced form on HPLC. Reduction with dithiothreitol followed by characterization on FPLC suggested that two thioethanol groups were covalently bound (via disulfide bonds) per SlOOb dimer. Furthermore, in good agreement, titration with DTNB suggested that the two remaining SH groups were free. All chemicals were high-grade, commercial, purified reagents. ‘“CaCl, (9.1 Ci/g) was from Amersham while ‘“ZnCl, (4.4 Ci/ g) was from LMRI, Gif/Yvette.

Methods Ion remounl from SlOOb proteins. Prior to binding experiments or fluorimetric titrations, ions (essentially Ca”‘) were removed from the proteins by precipitation with trichloroacetic acid (19). Absorption and fluorescence measurements. Absorption spectra were recorded on a Cary 219 spectrophotometer. Scattered light was corrected for as described (20). Fluorescence spectra were obtained with a PerkinElmer MPF44A spectrofluorometer interfaced with a Perkin-Elmer 7500 computer. Protein emission spectra were corrected for Raman emission by subtracting the buffer emission spectrum. Emission quantum yields were determined, taking L-Tyr in water as a reference (Tyr quantum yield = 0.14 at 20°C (21)). Fluorimetric titrations were performed by adding aliquots of CaClz or ZnCl, stock solution and monitoring the fluorescence intensity changes at the maximum emission wavelength. F/ou~ dial,vsis rxpwiments. The flow dialysis cell was as described (22), with a lower chamber (volume less than 0.1 ml) separated from the upper chamber by a Spectra Por (cutoff, 6000-8000) dialysis membrane. The upper chamber contained the protein in solution (1 ml) in the working buffer plus either 9.2 pM %aC12 or 2 pM “‘ZnCl,. The lower chamber was perfused with the same bufIer at 3.7 ml/min, using a Masterflex peristaltic pump. Fractions were collected every 24 s and aliquots of concentrated CaC12 or ZnS04 solutions were added to the upper chamber every five fractions. The last three fractions of each addition were counted in Aquasol (NEN) and the mean of these three values was used for calculations. The final chase-out was done with 10 mM CaCl, or 1 mM ZnSO, I)atn annlvsis. Binding data were analyzed according to Baudier et al. (4) and were further corrected for Ca”’ or Zn” binding to the dialysis membrane (23), for ligand leakage from the upper chamber during the experiment, and for upper chamber dilution by the addition of ligand aliquots. The corrected values of I, the average number of moles of ligand bound per mole of protein, and r, the free concentration of

least bind-

K,xf2K,K2xZf . +nK,K,...K,n” ‘=1+K,xfK,Kgx2+...+K,KL...Knzn’

[II

Fluorescence titrations were analyzed in relation to binding parameters, as previously described (25). In short, the fluorescence signal S, monitored as a function of the added ligand, was expressed as: S=S,,+S,K,x+S,K,K~n”+ . ..+S.K,Kz...K,r” l+K,x+K,K,r’+...+K,K,...K,x”

’

121

RESULTS

Structural

Properties of the Mixed Disulfide

Derivative

The mixed disulfide derivative of SlOOb protein and 2,2’-dithioethanol (MDS100b4) was indistinguishable from its reduced counterpart in amino acid composition, SDS-PAGE and gel filtration patterns, tryptic digestion maps, reactivity with SlOO/3 antibody in radioimmunoassay, or Western blot and activation of a potential target enzyme: Fru-1,6-bisphosphate aldolase (data not shown). Taken together these data strongly suggest that both proteins had similar structures. However, subtle differences could be detected. For example, in absorption spectra since apo-MDSlOOb had spectral features (smoothing of the 262-nm shoulder and decrease in the 26%nm peak) nearly identical to those obtained for the Ca2+- or Zn2’-loaded reduced protein (Fig. 1). Differences between the Tyr environments of the two proteins were confirmed by fluorescence data, under nondenaturating conditions, either in the absence or in the presence of Ca2+ or Zn2+ ions (Table I). Reduced protein quantum yields should be considered as refinements of those previously reported (5) since the very low emission spectra were measured with a much more accurate spectrofluorometer. Finally, conformational differences could also be inferred from tryptic digestion maps which were qualitatively similar but differed quantitatively, MDSlOOb being more sensitive to tryptic digestion (essentially in the apo-form) than the reduced protein. 4 Abbreviations used: MDSlOOb, mixed disulfide derivative of SlOOb protein and 2,2’-dithioethanol; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; TNS, 2-p-toluidinylnaphtalene6 sulfonate; DTNB, 5,5’-dithiobis (2.nitrobenzoic acid); FPLC, fast protein liquid chromatography.

176

MELY

AND

GERARD

conditions, four Zn” ions could bind to both proteins, and the affinity of reduced SlOOb protein seemed to be about twice that of MDSlOOb. In both proteins, the experimental points fitted adequately with models of four identical and independent sites (Fig. 3). Conformational

240

260

260

Wavelength

300

320

(nm)

FIG. 1. Absorption spectra of reduced SlOOb protein (a, b) and MDSlOOb (c). Spectra were recorded either in the absence of ions (a, c) or in the presence of 1 mM Ca”+ (b) for three different protein concentrations. For the sake of clarity, the Zn’+-loaded reduced species (superimposable on b) and the ion-loaded MDSlOOb species (similar to c) are not shown.

Ion-Binding Properties of the Mixed Disulfide Derivative: Comparison with Reduced Protein Ca’+-binding properties. The Ca2+-binding properties of both proteins were studied in 50 mM Tris, pH 7.8, to ensure sufficient ionic strength and good buffering potential. Results are summarized in Table II. The affinity of reduced SlOOb protein for Ca2+, in the absence of other ions, appeared fairly weak and, as in the case of calmodulin (26), the experimental points could be fitted, within the limits of experimental error, using a model of four identical and independent sites (Fig. 2A). In contrast, MDSlOOb, whose Cazf affinity was about four times greater than that of the reduced protein, was characterized by clearly positive cooperativity, as can be judged from the values of K2 and K3 compared to K, , the steepness of the binding curve and the poor fit with a model of four identical and independent sites (Fig. 2B). The presence of 120 mM K+ and 5 mM Mg2+ greatly reduced the affinity of native SlOOb protein for Ca2+ (two sites even became untitratable), but only slightly reduced the Ca2+-binding properties of MDSlOOb (all four sites remained titratable, with persistent positive cooperativity). Unlike K+ and Mg2’, the binding of Zn2+ enhanced the Ca2+-binding affinities, which became identical in both proteins. This effect of Zn2+ on Ca2+ binding was decreased to the same extent, in both proteins, by the presence of K’ and Mg2’. .Zr?+-binding properties. The Zn” -binding constants, summarized in Table III, were only determined in the presence of 120 mM K+ and 5 mM Mg2’ since our 6’ZnC12 specific activity was too low for accurate investigation of the high Zn2+ affinities previously described, at low ionic strength (4). Moreover, these physiologic concentrations of K+ and Mg2+ are likely to suppress the reported nonspecific Zn2+ -binding sites (4). Under these

Changes Induced by Ion Binding

Changes associated with Ca2’ binding. These changes were monitored both in the presence and in the absence of K+ and Mg2+, by fluorescence spectroscopy, using either (i) intrinsic Tyr 17 fluorescence (which is an a priori label of the N-terminal, Ca2+-binding EF hand) or (ii) TNS, a hydrophobic fluorescent label which enables monitoring of the exposure of hydrophobic domains. In the latter experiments, the association constants of both SlOOb species for TNS, determined by equilibrium dialysis, were lower than lo4 M-‘; thus TNS.SlOOb complexes were negligible and could not perturb the equilibrium between the various SlOOb.Cai (i = 0 to 4) species. Titrating the intrinsic fluorescence of reduced SlOOb protein with Ca2+, in the absence of other ions (Fig. 4A), showed that the changes in Tyr environment did not correlate with the binding isotherm but were parallel to the appearance of SlOOb species bound to at least two Ca2+. In the case of MDSlOOb, the titration curve did not clearly plateau: a small nonsaturable component appeared at high concentrations and hindered the determination of the specific conformational changes associated to the different MDS1OOb.Cai (i = 1 to 4) species. Nonetheless, since this component seemed rather small in comparison with the specific changes, it could be concluded that the titration curve correlated fairly well with the binding isotherm (Fig. 4B) and that the main conformational changes were associated with the appearance of MDS100b.Ca2 species. Under the same conditions, TNS titration curves for both proteins did not plateau since a major nonsaturable fluorescence change appeared (Fig. 5). Thus, no correlation was possible with the binding curves, however, it

TABLE Emission

Conditions” No ions 1 mM

0.5 mM 8

CC?’ %I’+

guanidinium chloride

I

Yields of Reduced SlOOb Protein and MDSlOOb

Quantum

Reduced protein

MDSlOOb

1.2 (kO.1) x lo-” (7) 8 (21.5) X lo-” (5) 5.3 (kO.7) x lo-” (3)

9.6 (kO.6) X lo-” (10) 6 (21) X 10 ~‘I(6) 2.9 (kO.4) x 10 a (4)

3.7 (fO.l)

3.7 (kO.2) x 10 2 (2)

M

x lo-’

(3)

” All protein solutions were in 50 mM Tris, pH 7.8. Excitation wavelength was 280 nm. Emission spectra were recorded and corrected as described under Experimental Procedures. Results are expressed as means t SEM; the number of samples is given in parentheses.

PROPERTIES

OF AN SlOOb PROTEIN TABLE

MIXED

177

DISULFIDE

II

Macroscopic Ca’+-Binding Constants of Reduced SlOOb and MDSlOOb, under Various Ionic Conditions SlOOb

Conditions”

Red’ MD Red MD Red MD Red MD

No ions 120

mM

KCI, 5

mM

K,

MgCl,

4 Zn”/SlOOb 4 Zn2’/S100b 120 mM KCI, 5 mM MgCl,

K2 (mM-‘)

(mM-I)

32 50 0.9 4 470 390 46 39

(23) (&lo) (10.1) (kl) (k50) (k40) (35) (k2)

1:‘; % 0.18 33 150 190 32 27

K,

(+-30) (*2) (kO.06) (-tlO) (k30) (k20) (k4)

(mM

K4 (mM-

‘)

451.4 (k9) (k0.6) 1 .o (f0.2) 130 (f20) 77 (&lo) 8.4 (kO.8) 9.1 (kO.7)

Ck2)

itb

‘)

22 10

(k9)

1.9 33 32 1.3 1.5

(f0.3) (k3) (?3) (kO.3) (fO.l)

(mM-‘)

40 11 (k2) (f10) 4 130 117 11 11

(?l) (?20) (*lo) (k2) (kl)

’ Experimental conditions were as described in Fig. 2. ’ k is the mean association constant given by I? = (K,K,K,K, )“4. ’ For each ionic condition, binding constants given for reduced SlOOb protein and MDSlOOb are expressed as means (k standard deviation). Experiments, made at least in duplicate, were found to be fairly reproducible. Binding experiments, experimental point correction, and data analysis were performed as reported under Experimental Procedures. In each case, the experimental points clearly plateau at four ions per where no plateau was reached) and could be fitted to the dimer (except for the reduced protein in the presence of 120 mM KCl, 5 mM M&l, general Adair-Klotz equation using four binding constants.

could be stated that (i) the apo-MDSlOOb species seemed slightly more hydrophobic than the apo-reduced form, (ii) Ca2+ induced only small TNS fluorescence enhancements for both proteins; these changes being more pronounced for the reduced protein and, (iii) the hydrophobic exposure increased monotically for reduced protein while at least t,wo steps appeared for MDSlOOb (as revealed by a plateau between lo-’ and 10 ~4M free Ca2+ concentration). In the presence of Kt and Mg’+, the intrinsic fluorescence titrations revealed, for the reduced protein, a clear correlation between the fluorescence changes and the appearance of protein species containing at least one Ca” (Fig. 6A), while for MDSlOOb, the changes were correlated rather with the appearance of MDSlOOb.Ca, and MDSlOOb.Ca,, species, each species contributing to half the maximal changes (Fig. 6B).

7

6

5

-log(free

4

Ca*+)

3

2

Changes associated with Zn2+ binding. Intrinsic fluorescence titration curves, for reduced protein, indicated that changes in the Tyr environment were essentially associated with the appearance of S100b.Zn3 species. Moreover, the fluorescence signal only increased sharply once two Zn2+ ions had bound (Fig. 7A), which suggests that the binding sites related to Tyr fluorescence changes were of lower affinity than those which left Tyr unaffected. In sharp contrast, MDSlOOb fluorescence changes were correlated with MDSlOOb.Zn, and MDS100b.Zn2 species (each contributing about 50% of the total changes) and were completed when two Zn’+ had bound to the protein (Fig. 7A), which suggests that the changes in the Tyr environment were associated with high affinity sites in this protein. Titration in the presence of TNS showed that, as for the Tyr signal, the TNS fluorescence only increased

7

6

5

-log(free

4

3

2

Ca*+)

FIG. 2. Ca”+-binding isotherms of reduced SlOOb protein and MDSlOOb under various ionic conditions. Reduced SlOOb protein (A) and MDSlOOb (B) concentrations were 50-80 pM in 50 mM Tris pH, 7.8 (0); plus 120 mM KCl, 5 mM MgCl, (W); 4 Zn’+/SlOOb molar ratio (0); 4 Zn”+/SlOOb molar ratio, 120 mM KCl, 5 mM MgCl, (a). The solid lines were drawn using the binding constants reported in Table II. The dotted lines represent the theoretical curves fitting, by a nonlinear least square procedure assuming four identical and independent sites, to the experimental points obtained in 50 mM Tris, pH 7.8.

178

MELY TABLE

(mM-‘)

Reduced protein MDSlOOb

490 (k60) 360 (k20)

K2 (mM-‘)

180 (k30) 63 (t6)

& (mM-‘)

30 (k5) 29 (k4)

of

l?

K4 (mM

‘)

20 (k2) 7.5 (-t-l)

(mM-‘)

85 (&lo) 47 (k5)

Note. Experimental conditions were as described in Fig. 3. Binding experiments, data treatment, expression of binding constants and I? are as indicated in Table II. Experiments, made at least in triplicate, were found to be fairly reproducible. For both proteins, a clear plateau was obtained for four Zn2+ per mole of protein, so the experimental points could be fitted to the general Adair-Klotz equation using four binding constants.

once two Zn2+ had bound to the reduced protein (Fig. 7B). In the case of MDSlOOb, the exposure of hydrophobic domains was correlated essentially to MDSlOOb.Zn, and MDS1OOb.Znz species, was considerably less than for the reduced protein, and was complete for three Zn2’ bound to the protein (as against two for Tyr fluorescence). DISCUSSION

Structural

Properties of the Mixed Disulfide

GERARD

or Zn’+-loaded reduced protein or in SlOOb protein chemically modified by acetamide groups (6).

III

Macroscopic Zn”-Binding Constants Reduced SlOOb and MDSlOOb K,

AND

Derivative

MDSlOOb is characterized by the blockage of two out of four Cys per dimer by thioethanol groups.3 The modified Cys were probably those at position 84, since these residues were reportedly shown to be selectively exposed to the solvent, in the presence of Ca2+, where due to their high reactivities, they could specifically react with different thiol-specific reagents such as DTNB (5), iodoacetamide, acrylodan, bimane (6), N-ethyl [2,3-14C]maleimide (14), or N-(1-pyrenyl)iodoacetamide (7). MDSlOOb was similar to its reduced counterpart in amino acid composition, electrophoretic and gel filtration patterns, reactivity with antibodies, and activation of a putative target enzyme. Thus, the overall conformations of both proteins appeared largely similar, which suggests that introducing the thioethanol groups does not drastically modify the protein structure. However, small differences may exist, as was revealed by the changes in the aromatic amino acid environments and the quantitative differences in tryptic digestion maps. Since the single Tyr and almost all the tryptic cleavage sites were located in the N-terminal part of the p subunit whereas both Cys were located in the C-terminal part (Fig. 8), it could be inferred that small conformational differences probably extended to the whole protein structure. The spectral modifications observed were not specific to the introduction of thioethanol groups since identical absorption spectra could be generated in Ca’+-

Ion-Binding Properties and Ion-Induced Conformational Changes Ca”’ binding to reduced SlOObprotein. In accordance with previous reports (4), reduced SlOOb protein binds four Ca” ions per dimer with rather low affinities. Our binding constants were even lower than those reported (4), probably because our experiments were performed at higher ionic strength (37 mM versus only 17 mM) and were corrected for nonspecific binding to the dialysis membrane. This nonspecific binding interference was especially large at high Ca2+ concentrations (representing between 20 and 50% of the total Ca2+ bound between 10e4 and lo-” M for reduced protein in 50 mM Tris, pH 7.8) and probably explains the existence of the four to five additional low-affinity Ca2+-binding sites previously reported (4). The binding isotherm was fairly close to the binding curve generated, assuming four identical and independent sites. Identical-shaped binding curves have already been reported for other Ca’+-binding proteins, such as calbindin 9K or calmodulin. The binding characteristics of the latter proteins have been widely studied but their binding mechanisms are still much debated. Indeed, for calbindin 9K, the closest-related protein to the SlOOp subunit, it has been suggested that Ca2+ binding involves either two nearly identical and independent sites (27) or two interactive sites with a twofold difference in affinity and with strong positive cooperativity (28). In the same way, several Ca2+ binding models have been proposed for calmodulin, with (i) four identical and independent sites (26), (ii) two pairs of cooperative sites (one low affinity and the other high) with strong positive cooperativity

1

7

6

5

-log(free

4

3

Zn2+)

FIG. 3. Zn”-binding isotherms of reduced SlOOb and MDSlOOb. Reduced SlOOb protein (0) and MDSlOOb (a) concentrations were 20-30 pM in 50 mM Tris, pH 7.8,120 miv KCl, 5 mM MgCl,. Solid lines were drawn using the binding constants reported in Table III. Dotted lines represent the theoretical curves (obtained assuming four identical and independent sites, as in Fig. 2) fitting the experimental points of both proteins.

PROPERTIES

OF AN SlOOb PROTEIN

a~80 2 fi 60 $ 3$ 40 ; 20 a 0

a, E g f$ b 1 ; Oc 5

4

-log(free

-

3

Ca*+)

MIXED

179

DISULFIDE

80 60 40 20 0 6

5

-log(free

4

3

Ca*+)

FIG. 4. Tyrosine fluorescence titration of reduced SlOOb and MDSlOOb as a function of free Ca”. Reduced SlOOb (A) or MDSlOOb (B) (15 Procedures. Excitation to 25 FM) in 50 mM Tris, pH 7.8, was titrated with increasing amounts of Ca’+, as indicated under Experimental wavelength was 280 nm. Fluorescence intensities (0) measured at 303 nm for both proteins were taken as 0% for calcium-free protein and 100% in 1 mM CaCl,. The solid lines were calculated from Eq. [2], defined under Experimental Procedures. The dotted lines in (A) and (B) represent respectively the appearance of the SlOOb. Ca, (; = 1 to 4) species (for the reduced protein) and the binding isotherm of MDSlOOb, computed by using the binding constants reported in Table II.

between the sites of each pair, (29) or (iii) four sequential and ordered binding sites (19). Binding experiments alone are not sufficient to discriminate between different binding models (30) and other techniques are needed to provide additional information. In the case of SlOOb protein, Tyr-intrinsic fluorescence seemed well-suited for this purpose since there is only one Tyr per monomer, located at position 17 in the N-terminal EF hand, and could thus probe, a priori, the filling of this site. The titration of Tyr fluorescence did not parallel the binding isotherm but was rather associated with the formation of S1OOb.Caz species, which suggests that the four binding sites are not equivalent unless we adopt the hypothesis of Cox et al. (31) on “disproportionate” conformational changes (i.e., that the

4

l ---

il 6

5

-log(free

4

3

Ca*+ I

FIG. 5. TNS fluorescence titration of reduced SlOOb and MDSlOOb as a function of free Ca’+. TNS (10 pM) and 25 pM reduced SlOOb (0) or MDSlOOb (X) were titrated in 50 mM Tris, pH 7.8, with increasing Ca”+ concentrations as described under Experimental Procedures. Excitation wavelength was 360 nm and fluorescence intensities were measured at 437 nm (where the emission was maximum for the Ca’+loaded species). The straight baseline represents the fluorescence of 10 fiM TNS in the same buffer without protein.

changes are associated with the formation of S1OOb.Caz whatever the Ca2+ sites occupied). Nor could the model of paired cooperative sites (29) be applied since the symmetrical structure of the apo-SlOOb protein, deduced from ‘H NMR data (3, 32), could hardly be reconciled with the existence of low and high affinity pairs of binding sites. A model of two identical pairs of cooperative sites would be more realistic but should lead to a somewhat steeper binding curve. Finally, the model of sequential binding was the most tempting one; it would imply that the second Ca2+ binds to an N-terminal Ca’+binding site. However, not enough data were available to accredit this model and alternative models can obviously not be ruled out. As already reported (3,4), a dramatic decrease in Ca2+ affinity was observed in the presence of K+ and Mg”+. Only two Ca’+-binding sites per dimer remained titratable but their saturation led to Tyr conformation changes identical to those in the absence of K+ and I$?+, confirming that the two C-terminal binding sites are probably most affected by the presence of K’ and Mg2+ (4). A rather intriguing point in titrations performed either in the absence or in the presence of Ki and Mg2+ was the fact that all the conformational changes were associated with the appearance of only one Ca’+-bound species despite the probable symmetric dimeric protein structure of the apo-protein, which suggests some asymmetrical behavior during Ca2+ binding. It would be tempting to deduce that the binding of Ca2+ to one Nterminal EF hand of the dimer modifies not only its own Tyr environment but also that of the other N-terminal site which becomes insensitive to further Ca” binding. Other hypotheses are of course possible and so, further experiments are called for.

180

MELY

AND

GERARD

100 80 60 40 20 0

-log(free

Ca*+)

-log(free

Ca*+)

FIG. 6. Tyrosine fluorescence titration of reduced SlOOb and MDSlOOb, in the presence of K’ and Mg’+, as a function of free Ca”’ . Reduced SlOOb (A) or MDSlOOb (B) (20 to 25 FM) in 50 mM Tris, pH 7.8, was titrated with increasing amounts of Ca*‘, in the presence of 120 mM KC1 and 5 mM MgCl,, as indicated under Experimental Procedures. The representation of the signal (0) and the meaning of both solid and dotted lines are as in Fig. 4A. It should be noted that in both proteins the absolute fluorescence changes between the apo- and the Ca’+-loaded forms were identical to those obtained in the absence of K+ and Mg’+.

Since Ca” binding to the mixed disulfide derivative. the K, macroscopic binding constant was similar to that obtained for reduced protein, it could be inferred that introducing thioethanol groups probably did not greatly modify the apo-protein Ca”+ affinities but rather induced different conformational changes after the binding of the first Ca2+, enabling strong positive cooperativity for the following Ca2+ to bind. Interestingly, as in the reduced protein the main conformational changes were associated with MDS100b.Ca2 species, which suggests that although the binding constants were markedly changed, the binding sequence might be unaffected. Thus, the cooperativity observed probably corresponded merely to an increase in already existing interactions between two or more binding sites. The most striking effects of the chemical changes on Cys residues were observed upon Ca2+ binding in the

presence of K’ and Mg2+, since all four sites remained titratable (with an even higher positive cooperativity for the second Ca2+ to bind), which suggests a marked decrease in the effect of K+ and Mg2+ on Ca2+ binding to the C-terminal EF hands. While the final Ca*+-loaded MDSlOOb structure was probably similar to that observed in the absence of K’ and Mg2+ (the decrease in Tyr fluorescence being the same), the binding sequence appeared somewhat different, since the changes in the Tyr environments were associated essentially with the appearance of MDSlOOb.Ca, and MDS100b.Ca3 species and were, thus, uncoupled. Flow dialysis Zn2+ binding to reduced SlOObprotein. experiments performed in the presence of 120 mM Kt and 5 mM Mg2+ suggested that four Zn2+ ions could bind to reduced SlOOb protein with a much higher affinity than Ca’+. Two supplementary binding sites and higher

; 2.5 50 I $ L g 9 g I f O

1

2

Bound Zn2+/mol

3 s100b

4

2.0 1.5 1.0 0.5 0.0 0

1

2

3

4

Bound Zn 2+/mol slOOb

FIG, 7. Tyrosine and TNS fluorescence titrations of reduced SlOOb and MDSlOOb as a function of bound Zn*+. Reduced SlOOb (0) or MDSlOOb (X) (10 pM) in 50 mM Tris, pH 7.8, 120 mM K+, 5 mM Mg2+ was titrated with increasing amounts of Zn2+ as described under Experimental Procedures. Tyr fluorescence signal (A), excited at 280 nm, was monitored at 303 nm and is taken as 0% for ion-free species and 100% in the presence of 0.5 mM ZnCl,. TNS fluorescence signal (B), excited at 360 nm, was monitored at 430 nm (where the fluorescence intensity was maximal for the Zn “-loaded species). TNS fluorescence is represented in relative units since no plateau could be obtained for the reduced protein. Solid lines were calculated from Eq. [2] and are represented as functions of moles of Zn”+ bound per mole of SlOOb protein.

PROPERTIES

OF AN SlOOb PROTEIN

His25 His1 5 . A

MIXED

DISUI,FIDE

181

His42

FIG. 8. Schematic representation of’ SlOOg .su b unit. SlOOfi subunit polypeptide backbone is shown as a solid line, with the positions of individual amino acids (Cys, Tyr, and His) indicated relative to the two Ca*+-binding sites. Each Ca2’ -binding EF hand comprises a central loop surrounded by two (Y helices. It should be borne in mind that the C-terminal binding EF hand is a “classical” one while the N-terminal binding EF hand constitutes a “pseudo” EF hand with two supplementary amino acids.

binding constants have been reported under similar conditions (4): these differences could stem, as in Ca2+ binding, from buffer condition differences and from nonspecific binding due to the dialysis membrane. As for Ca’+, the Zn”’ -binding isotherm is close to the binding curve generated, assuming four identical and independent sites. However, this mechanism was largely ruled out by the Tyr fluorescence changes which only appeared once two Zn” had bound, which suggests that two classes of Zn’+-binding sites probably exist: a high affinity one giving no fluorescence changes and a low affinity one associated with changes in the Tyr environment. Moreover, the fluorescence signal was essentially associated with the appearance of only one species: S100b.Zn3, which suggests, as in the case of Ca”‘, some asymmetrical behavior with possible interaction between sites. The existence of two classes of Zn’+-binding sites was confirmed by TNS fluorescence data, since the TNS signal also increased only once two Zn2+ had bound to the protein, which suggests that the hydrophobic domain exposure was associated with the low affinity sites. Since Zn2+ binding to the high affinity sites did not markedly affect the Tyr environment, it is tempting to assume that these sites are located primarily in the Cterminal part of the molecule, as has already been suggested (4). In contrast, the high His content (at positions 15, 25, and 42) and the Tyr conformational changes make the N-terminal part a likely candidate for the lowaffinity binding sites. BindZn’+ binding to the mixed disulfide derivative. ing experiments suggested that MDSlOOb could bind four Zr?+ with an affinity two times lower than the reduced protein. As in reduced protein, intrinsic and TNS fluorescence titrations revealed the probable existence of two classes of sites, but in contrast to reduced protein, Tyr conformational changes occurred for the first two Zn”+ bound per dimer and hydrophobic domain exposure for the first three. Since the same Zn”+-binding sites were probably involved in both proteins, it could be tentatively concluded that the chemical changes on Cys residues reversed the Zn”+-binding sequence with regard to the reduced protein. In both proteins (in Zinc regulation of Ca2+ affinities. the presence or in the absence of K+ and Mg’+), Znzt binding markedly increased Ca”‘-binding affinities

which became identical in both proteins, which suggests that Zn2+ conferred similar conformations on their Ca”+binding sites. Furthermore, the strong positive cooperativity for Ca” binding to MDSlOOb vanished in the presence of Zn’+. Thus, it could be concluded that the were probably thioethanol groups in MDSlOOb “inactivated” in such a way that they could no longer interact with any Cazi -binding site. This Zn”+-induced increase in Ca” -binding affinities has already been reported by Baudier (33) who suggested that it might result from increased accessibility following dimer dissociation. This could not apply to our experiments, which were performed at protein concentrations well above previous reported dimer dissociation constants (15). This absence of dissociation was further confirmed by anisotropy measurements (data not shown). Conclusion Comparing the structural and ion-binding properties of reduced SlOOb protein with those of a model mixed disulfide derivative allowed us to draw following conclusions. (i) The introduction of thioethanol groups on two out of four Cys per dimer did not greatly modify the overall protein structure but increased largely Ca” affinities (essentially at physiological concentrations of Kf and Mgzl ) by inducing strong positive cooperativity. (ii) As in the mixed disulfide derivative, the binding of Ca2+ to reduced native SlOOb protein probably involves nonequivalent interacting sites. (iii) The two proteins bind four Znzi ions, in two distinct classes of sites and this binding leads to conformational changes giving rise to identical Ca’+-binding affinities for both proteins. The properties of this mixed disulfide derivative suggest that the modifications induced by the disulfide formation on the near environment of Cys 84 residues are a thermodynamically favorable event (especially in regard to Ca2+ binding) compatible with the physiological relevance of SlOOb protein mixed disulfide derivatives. When larger substitutents (e.g., proteins like 7 (2) proteins (15, 16)) are considered, the interactions between SlOOb protein and the substituent should be of course much more complex. So, further experiments on such

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derivatives are called for to progress in this interesting new field of investigation for SlOOb protein. On the other hand, the preliminary hypotheses elaborated here on Ca’+- and Znzt -binding mechanisms need to be confirmed and completed by additional information provided on either native or modified proteins to yield more definitive answers on the intriguing binding properties of SlOOb protein.

ACKNOWLEDGMENTS We thank Dr L. J. Van Eldik for performing the immunochemical and enzyme activation experiments, Dr J. Haiech for stimulating advice, and Mrs M. Wernert for expert editorial assistance.

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