Mg2+-switch model1

Article No. mb982116

J. Mol. Biol. (1998) 283, 679±694

Oligomerization and Divalent Ion Binding Properties of the S100P Protein: A Ca2‡/Mg2‡-switch Model Alexey V. Gribenko and George I. Makhatadze* Department of Chemistry and Biochemistry, Texas Tech University, Lubbock TX 79409-1061, USA

S100P is a 95 amino acid residue protein which belongs to the S100 family of proteins containing two putative EF-hand Ca2‡-binding motifs. In order to characterize conformational properties of S100P in the presence and absence of divalent cations (Ca2‡, Mg2‡ and Zn2‡) in solution, we have analyzed hydrodynamic and spectroscopic characteristics of wild-type and several variants (Y18F, Y88F and C85S) of S100P using equilibrium centrifugation, gel-®ltration chromatography, circular dichroism and ¯uorescence spectroscopies. Analysis of the experimental data shows the following. (1) In agreement with the predictions there are two Ca2‡-binding sites in the S100P molecule with different af®nity; the high af®nity binding site has an apparent binding constant of 107 Mÿ1 and the low af®nity binding site has an apparent binding constant of 104 Mÿ1. (2) The high and low af®nity Ca2‡-binding sites are located in the C and N-terminal parts of the S100P molecule, respectively. (3) These C and N-terminal sites can also bind other divalent ions. The C-terminal site binds Zn2‡ (with relatively low af®nity 103 Mÿ1), but not Mg2‡. The N-terminal site binds Mg2‡ with the apparent binding constant 102 Mÿ1. (4) Binding of Ca2‡ to the C-terminal site and binding of Mg2‡ to the N-terminal site occur in the physiological concentration range of these ions (micromolar for Ca2‡ and millimolar for Mg2‡). (5) Oligomerization state of the S100P molecule appears to change upon addition of Ca2‡. On the basis of these observations a plausible model for S100P as a Ca2‡/Mg2‡ switch has been proposed. # 1998 Academic Press

2‡

*Corresponding author

Keywords: EF-hand protein; Ca -binding; conformational changes; circular dichroism; ¯uorescence

Introduction Calcium ions serve as secondary messengers during the control of biochemical pathways. This control of cellular signal transduction is partly achieved through the interactions with Ca-binding Abbreviations used: CaBP, calcium binding protein; CD, circular dichroism spectroscopy; ANS, 8-anilino-1naphtalenesulfonic acid; DTNB, 5,50 -dithio-bis(2nitrobenzoic acid); pCa, negative logarithm of Ca2‡ concentration; pMg, negative logarithm of Mg2‡ concentration; pZn, negative logarithm of Zn2‡ concentration; WT-S100P, wild-type S100P protein; Y18F-S100P, S100P with the Tyr18 ! Phe amino acid substitution; Y88F-S100P, S100P with the Tyr88 ! Phe amino acid substitution; C85S-S100P, S100P with the Cys85 ! Ser amino acid substitution; 3D, threedimensional. E-mail address of the corresponding author: [email protected] 0022±2836/98/430679±16 $30.00/0

proteins (CaBP). A large class of CaBP share a common Ca-binding structural motif, the EF-hand (Kretsinger, 1976; Kawasaki & Kretsinger, 1995). Among the EF-hand proteins, the S100 family is possibly the largest (Schafer & Heizmann, 1996). These proteins consist of about 100 amino acid residues and have two EF-hand motifs in their primary structure (Hilt & Kligman, 1991; Schafer & Heizmann, 1996; Zimmer et al., 1995). About 20 different proteins belonging to the S100 family have been identi®ed so far. Although there is 25% to 60% sequence identity between different S100 proteins, they have diverse tissue and cellular localization as well as diverse function and metal binding speci®cities (Hilt & Kligman, 1991; Schafer & Heizmann, 1996). Variations in the expression levels in S100 proteins were demonstrated for a large number of neurological and cardiological human diseases and for tumor development: cardiomyopathies (S100A; Schafer & Heizmann, 1996), # 1998 Academic Press

680 Alzheimer's disease (S100B; Marshak, 1990; Marshak & Pena, 1992; Marshak et al., 1992), Down's syndrome (S100B; Allore et al., 1988), epilepsy (S100B; Grif®n et al., 1995), breast cancer (S100B; Lee et al., 1991, 1992), type I diabetes (S100B; Zimmer et al., 1997), metastasis (CAPL; Davies et al., 1993), psoriasis (psoriasin; Hoffmann et al., 1994) cystic ®brosis (MRP14; Renaud et al., 1994), melanoma (calcyclin; Weterman et al., 1993) and prostate cancer (S100P; Averboukh et al., 1996). Different expression and cellular localization of S100 proteins probably indicate their different functional role (Zimmer et al., 1995). Detailed studies of three proteins (calbindin D9k, S100B and calcyclin) of the S100 family have revealed particular structural properties. Calbindin D9k is a monomeric protein which can bind Ca2‡, Mg2‡ and Mn2‡ (Kordel et al., 1993; Andersson et al., 1997). In contrast, calcyclin (S100A6) is a homodimer which binds four Ca2‡ molecules per protein dimer (Potts et al., 1995, 1996). S100B is also a homodimer; however, its properties differ from that of calcyclin. In addition to Ca2‡, S100B also binds Zn2‡, with a binding af®nity that is considerably higher than that for Ca2‡ (Baudier et al., 1986; Baudier & Cole, 1989; Mani & Kay, 1987). Moreover, Ca2‡-binding to S100B leads to signi®cant structural perturbations (Drohat et al., 1998; Smith et al., 1996; Smith & Shaw, 1997; Matsumura et al., 1998). All of these show that each protein of the S100 family might be unique in its structural and metal binding properties re¯ecting their particular functional role. Human S100P, a 95 amino acid residue protein, was ®rst isolated several years ago (Emoto et al., 1992; Becker et al., 1992) and has 50% and 35% sequence identity with S100B and calcyclin, respectively (Figure 1). Recently, the difference in the regulations of expression of this protein in the androgen-dependent and androgen-independent

Ca2‡/Mg2‡-switch in S100P

prostate cancer cell lines has been demonstrated (Averboukh et al., 1996). In our previous paper (Gribenko et al., 1998) we have shown that the circular dichroism (CD), tyrosine and ANS ¯uorescence spectroscopies can be used to monitor conformational changes on the S100P molecule upon Ca2‡-binding. Sequence analysis and simpli®ed 3D-homology modeling of S100P based on the solution structures of apo-S100B (Drohat et al., 1996) and apo-calcyclin (Potts et al., 1995) reveals the presence of two putative Ca2‡-binding sites located in the N and C-terminal parts of the molecule. To facilitate the assignment of Ca2‡-binding sites, we have generated two amino acid substitutions in the sequence of S100P: tyrosine residues at positions 18 and 88 were substituted with phenylalanines (Figure 2). Tyrosine 18 is located very close in the sequence to the putative N-terminal Ca2‡-binding site. By mutating Tyr18 to Phe we expected to make this N-terminal Ca2‡-binding site ``invisible'' to ¯uorescence spectroscopy. Tyrosine 88 is located in the C terminus of the molecule and, as judged by the structures of S100B and calcyclin, is involved in the formation of the dimer interface (Potts et al., 1995, 1996; Drohat et al., 1996, 1998). In addition to these two amino acid substitutions, we have generated a third amino acid substitution in which cysteine (Cys) at position 85 was substituted by a serine (Ser) residue. The C-terminal cysteine is extremely conserved in the sequences of S100 proteins (Figure 1) so we replaced it with another small residue, Ser, to elucidate the effect of Cys85 on the conformational properties of S100P. Here we report the results of detailed studies of the effects of Ca2‡, Mg2‡, and Zn2‡ on the conformational properties of the S100P protein in solution. All four proteins (WT, Y18F, Y88F and C85S) were studied by ¯uorescence and circular dichroism spectroscopies, analytical cen-

Figure 1. Comparison of the sequence of S100P with the sequences of other S100P proteins. Highlighted are residues identical to those in S100P. The amino acid residues involved in the formation of helical segments of S100 proteins with known 3D structures are single-underlined (Svensson et al., 1992; Potts et al., 1995; Drohat et al., 1996, 1998; Kilby et al., 1996; Matsumura et al., 1998; Sastry et al., 1998; Smith & Shaw, 1998). Residues involved in the Ca2‡ coordination are indicated as * (mainchain carbonyl) or { (side-chain carboxylate). The tyrosine residues in positions 18 and 88, and the cysteine in position 85 of the S100P sequence are shown in bold. Pre®xes are as follows: b, bovine; h, human; r-rat.

681

Ca2‡/Mg2‡-switch in S100P

vations (Gribenko et al., 1998). This indicates that the overall fraction of amino acid residues involved in the formation of the regular elements of secondary structure (presumably four a-helices) of S100P is unaffected by Ca2‡. The results with S100P are consistent with the ®nding that Ca2‡ does not affect the secondary structure content of other EFhand proteins, e.g. calbindin D9k (Skelton et al., 1990a,b, 1995), calmodulin (e.g. see Finn et al., 1995), calcyclin (Potts et al., 1996; Sastry et al., 1998) or S100B (Drohat et al., 1996, 1998; Kilby et al., 1996; Smith et al., 1996; Smith & Shaw, 1997; Matsumura et al., 1998). Ca2‡ induces changes in the near-UV CD spectra of S100P Figure 2. Three-dimensional model of the structure of S100P dimer. The side-chains of Tyr18, Tyr88, Cys85 are shown in the CPK representation. Solely for the illustrative purposes of the positions of the putative Ca2‡-binding sites, two calcium molecules were superimposed on the structure and are shown as blue spheres. The model was built using the coordinates of apo-S100B (1SYM, Drohat et al., 1996) and the threading option of the Swiss PDB Viewer (http://www.pdb.bnl.gov/expasy/ spdbv/mainpage.html).

trifugation, and gel-®ltration chromatography. From these experiments we have derived a plausible ``Ca2‡/Mg2‡-switch'' model of the conformational behavior of S100P in solution under physiological conditions.

Results and Discussion Effects of C85S, Y18F, and Y88F amino acid substitutions on the secondary structure of S100P Secondary structure content for WT, C85S, Y18F, and Y88F forms of S100P was assessed from the comparison of the far-UV CD spectra (data not shown). The overall shape of the spectra was found to be similar for all four proteins. The spectra display minima at 222 and 209 nm is characteristic of proteins with high helical structure content (e.g. see Yang et al., 1986; Woody, 1995). The absolute values of the spectra for C85S, Y18F, and Y88F coincide within the experimental error (estimated standard deviation 7%), with the spectrum for the WT protein. This allows us to conclude that there are no dramatic changes in the global fold of S100P due to the amino acid substitutions at positions C85S, Y18F and Y88F. This result is expected because the amino acid substitutions do not dramatically change the nature of the side-chain in terms of size, shape or hydrophobicity. Addition of 5 mM Ca2‡ did not affect the far-UV CD spectra of the S100P proteins (data not shown), which is in agreement with our previous obser-

Near-UV CD spectra of the WT, C85S, Y18F and Y88F proteins are shown in Figure 3. In the absence of Ca2‡, all four proteins have spectra with minima at 262 and 268 nm. Minima at 262 and 268 nm are characteristic of a well packed and rigid environment of phenylalanine side-chains (Strickland, 1974; Woody, 1995; Pain, 1996). There are six phenylalanine residues in the sequence of WT-S100P and C85S, and seven in Y18F and Y88F proteins. At least some of these are involved in the formation of tertiary interactions. Spectra of apoproteins are also characterized by a relatively small ellipticity in the 275 to 290 nm range. Ellipticities at these wavelengths are usually associated with tyrosine and/or tryptophan residues (e.g. see Pain, 1996). S100P contains no tryptophan residues, therefore only tyrosine residues will be the major contributors to the ellipticity in the 275 to 290 nm range. When both positions 18 and 88 are tyrosine (WT and C85S), the ellipticity in the 275 to 290 nm range is negative. The ellipticity is also negative in this wavelength range for Y88F-S100P, whereas the ellipticity for Y18F-S100P is small but positive. Small values for ellipticity in the 275 to 290 nm range usually indicate that tyrosine residues are relatively solvent exposed and their side-chains are not involved in tertiary interactions (Pain, 1996). Addition of Ca2‡ induced notable perturbation of the near-UV CD spectra of S100P. These changes are particularly signi®cant in magnitude for the WT, C85S and Y88F proteins. For these proteins, a small negative ellipticity in the 275 to 290 nm range observed for apo-forms became large and positive in the presence of 5 mM Ca2‡. In contrast, changes in the near-UV CD signal for Y18F protein decreased from a small positive ellipticity to a small negative ellipticity at increased Ca2‡ concentrations. For all four proteins, the changes in the near-UV CD spectra indicate a signi®cant structural rearrangements in the tertiary structures upon addition of Ca2‡. These structural changes do not have a signi®cant effect on the far-UV CD spectra, i.e. addition of Ca2‡ affects the relative orientation of the helical regions in the protein without affecting the relative number of amino acid residues involved in the formation of these helices.

682

Ca2‡/Mg2‡-switch in S100P

Figure 3. Changes in the near UV-CD spectra as a function of concentration of Ca2‡ in buffer A for WT-S100P (pCa ˆ 1, 8.69, 8.00, 7.47, 7.04, 6.44, 5.92, 5.25, 4.53, 4.04, 3.63, 3.04, 2.03), C85S-S100P (pCa ˆ 1, 8.69, 8.00, 7.47, 7.01, 6.44, 5.92, 5.25, 4.53, 4.04, 3.63, 3.04, 2.03), Y18F-S100P (pCa ˆ 1, 8.35, 7.87, 7.41, 6.98, 6.13, 5.00, 4.43, 3.67, 2.05), and Y88F-S100P (pCa ˆ 1, 8.69, 8.00, 7.47, 7.01, 6.44, 5.92, 5.25, 4.53, 3.63, 3.04, 2.03). Arrows indicate the direction of change upon an increase in the concentration of Ca2‡.

For all four proteins, the changes that occur in the near-UV CD spectra upon addition of Ca2‡ represent titration curves. Figure 4 shows the dependence of the changes in ellipticity at 262, 268 and 276 nm on the concentration of Ca2‡. The wavelengths selected sample different parts of the near-UV CD where the signal orig-

inates from different amino acid residues and, possibly, from different parts of the structure. For the WT, C85S and Y88F S100P, changes in ellipticity at all three wavelengths were biphasic and concurrent (Figure 4). In contrast, the Y18F protein shows a single transition at pCa  6 to 8.

Figure 4. Changes in the ellipticity of WT-S100P, C85S-S100P, Y18F-S100P and Y88F-S100P at different wavelengths (*, 262 nm; &, 268 nm; ~, 276 nm) as a function of concentration of Ca2‡ in buffer A. Lines represent the ®t of the experimental data to equations (5) or (6) using parameters listed in Table 1.

683

Ca2‡/Mg2‡-switch in S100P

Table 1. Binding parameters (apparent binding constants, K1 and K2; maximal changes in the spectroscopic probe, Q1 and Q2) for Ca2‡ to S100P proteins obtained by ®t of experimental data to equations (5) and (6) Solvent WT

C85S

Y18F

Y88F

0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 5 mM MgCl2 5 mM MgCl2 50 mM MgCl2 50 mM MgCl2 100 mM MgCl2 100 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 5 mM MgCl2 5 mM MgCl2 50 mM MgCl2 50 mM MgCl2 100 mM MgCl2 100 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 5 mM MgCl2 5 mM MgCl2 50 mM MgCl2 50 mM MgCl2 100 mM MgCl2 100 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 0 mM MgCl2 5 mM MgCl2 5 mM MgCl2 50 mM MgCl2 50 mM MgCl2 100 mM MgCl2 100 mM MgCl2

Method Tyr ANS CD262 nm CD268 nm CD276 nm Tyr ANS Tyr ANS Tyr ANS Tyr ANS CD262 nm CD268 nm CD276 nm Tyr ANS Tyr ANS Tyr ANS Tyr ANS CD276 nm Tyr ANS Tyr ANS Tyr ANS Tyr ANS CD262 nm CD268 nm CD276 nm Tyr ANS Tyr ANS Tyr ANS

K1 (Mÿ1) 6

8.7
10 1.1
107 5.5
107 2.9
107 2.6
107 5.0
106 1.4
107 2.2
107 3.9
106 1.3
107 2.2
106 1.6
107 1.1
107 3.3
107 3.0
107 2.8
107 9.0
107 1.5
108 9.2
106 1.1
107 1.0
107 3.2
106 5.1
107 1.7
106 1.3
107 6.1
107 3.0
107 7.5
106 2.0
106 4.3
106 1.1
106 2.0
106 2.6
106 2.9
107 3.2
107 2.9
107 1.6
107 1.9
108 3.3
106 3.4
106 1.5
106 1.6
106

Q1 ÿ2 91 6 10 10 ÿ7 78 ÿ4 60 ÿ3 40 13 93 10 16 20 12 87 11 73 10 62 ÿ15 80 16 ÿ17 46 ÿ12 47 ÿ10 37 ÿ23 54 5 9 12 ÿ32 80 ÿ22 67 ÿ18 51

K2 (Mÿ1) 4

1.6
10 2
104 2.6
103 2.8
103 2.9
103 4.6
103 1.9
104 3.2
103 1.1
104 1.6
103 4.5
103 9.0
103 5.0
103 4.0
103 5.0
103 4.4
103 6.6
103 8.9
103 1.4
104 3.0
104 1.0
104 1.4
104 ± 1.5
103 ± ± 2.1
103 ± 5.0
102 ± 4.0
102 2.2
104 2.7
104 4.4
103 4.3
103 4.2
103 6.5
103 1.3
104 3.5
103 6.0
103 2.7
103 5.0
103

Q2 ÿ15 9 25 43 45 ÿ13 21 ÿ17 50 ÿ17 80 ÿ5 7 20 36 45 ÿ4 14 ÿ4 27 ÿ2 38 ± 24 ± ± 54 ± 62 ± 71 ÿ25 49 11 20 25 ÿ16 21 ÿ23 46 ÿ28 58

Units are % for the Tyr and ANS ¯uorescence and mdeg
cmÿ2 dmolÿ1 for near-UV CD experiments.

Changes in the ellipticity of near-UV CD spectra upon addition of Ca2‡ were analyzed according to equations (5) or (6) in order to estimate the apparent binding constant for Ca2‡. Comparison of the ®tted isotherms with the experimental results are shown in Figure 4. Table 1 presents the values for the apparent equilibrium constants estimated from the ®t. As expected from sequence analysis there are two Ca2‡-binding sites in the S100P molecule; a high af®nity Ca2‡-binding site with an apparent binding constant of 107 Mÿ1 and a low af®nity Ca2‡-binding site with an apparent binding constant of 104 Mÿ1. The low af®nity site is located in the N-terminal part of the molecule, as follows from the lack of the low af®nity transition in Y18F protein. Y18 is located in close proximity to the putative N-terminal Ca2‡-binding site. Thus, the N-terminal low af®nity binding site is either ``invisible'' in the near-UV CD or is rendered unable to bind Ca2‡ as a result of Y18F substitution. In either case, it is reasonable to suggest that the N-terminal

Ca2‡-binding site is the low af®nity binding site. Additional evidence for this conclusion follows from the Tyr ¯uorescence spectroscopy data. Ca2‡ induces changes in the Tyr fluorescence of S100P Figure 5 shows Ca2‡-induced changes in the Tyr ¯uorescence of S100P. Addition of Ca2‡ quenches ¯uorescence intensity for WT protein in a biphasic manner. Analysis of the data in terms of two independent binding sites (equation (6)) gives estimates for the apparent equilibrium binding constants similar to those obtained from the analysis of nearUV CD data (Table 1): 107 Mÿ1 for the high af®nity Ca2‡-binding site and 104 Mÿ1 for the low af®nity Ca2‡-binding site. Similarity of the apparent equilibrium constants for Ca2‡ obtained by two different spectroscopic methods provides strong evidence that changes in the S100P structure upon

684

Ca2‡/Mg2‡-switch in S100P

Figure 5. Changes in the Tyr ¯uorescence intensity as a function of concentration of Ca2‡ in buffer A for WT-S100P, C85S-S100P, Y18F-S100P and Y88F-S100P. Filled symbols are experimental results obtained at 0 mM (black), 5 mM (red), 50 mM (blue) and 100 mM (green) concentrations of Mg2‡. Lines represent the ®t of the experimental data to equations (5) or (6) using parameters listed in Table 1.

the addition of Ca2‡ are indeed related to Ca2‡binding. The effect of Ca2‡ on the Tyr ¯uorescence of Y88F-S100P is comparable to that observed for WT-S100P (Figure 5); biphasic transition with similar apparent binding constants (Table 1). In this variant of S100P, only one tyrosine residue, Y18, is present, indicating that this tyrosine residue changes conformation upon Ca2‡-binding to both low and high af®nity binding sites. In contrast, Y18F-S100P shows monophasic transition (Figure 5). This result is similar to the change observed upon Ca2‡ titration of Y18F monitored by near-UV CD spectroscopy (Figure 4). Analysis of the Ca2‡ titration curve monitored by Tyr ¯uorescence for Y18F-S100P yields an apparent binding constant similar to that of the high af®nity binding site (Table 1). It is also similar to the apparent binding constant for the high af®nity site for WT and Y88F proteins estimated from the Tyr ¯uorescence data (Table 1). This result again indicates that the low af®nity Ca2‡-binding site is located in the N-terminal part of the S100P molecule. However, as in the case of near-UV CD spectroscopy we can consider two possible reasons for the absence of the low af®nity transition in the Y18F protein: the changes in Tyr ¯uorescence cannot follow the Ca2‡-binding due to the absence of the Y18 spectral probe or the Y18F substitution affects the structure of the N-terminal Ca2‡-binding site and this site no longer binds Ca2‡. Changes in the Tyr ¯uorescence of the C85S protein are distinct from those of the other three S100P proteins. In the case of WT, Y18F and Y88F, Ca2‡

quenches the protein Tyr ¯uorescence. In contrast, the Tyr ¯uorescence for C85S-S100P is initially increased by increasing the Ca2‡ concentration up to pCa  6. At pCa > 6 the Tyr ¯uorescence intensity decreases. Analysis of the data according to equation (6) shows that in spite of the difference in the overall shape of the Ca2‡ titration pro®le, the apparent equilibrium constants, for both high and low af®nity sites, are on the order of 107 Mÿ1 and 104 Mÿ1, respectively. These values are similar to those obtained from near-UV CD data and are similar to those of WT, Y88F and Y18F (Table 1). Thus, the difference in the shape of the Tyr ¯uorescence pro®le upon Ca2‡ titration for the C85S protein is not directly linked to the ability of this protein to bind Ca2‡. It appears that the distinct Ca2‡ titration Tyr ¯uorescence pro®le for C85S is related to a difference in the oligomerization properties of this variant (see discussion below). Both near-UV CD and Tyr ¯uorescence spectroscopies monitor Ca2‡-induced changes in the S100P protein by following changes in the spectral properties of internal probes. The use of an external ¯uorescence probe can provide more information about the global properties of the molecule. ANS ¯uorescence can serve as one such probe for Ca2‡-induced changes in S100P (Gribenko et al., 1998). Ca2‡ induce changes in the ANS fluorescence of S100P We have shown that the changes in the ANS ¯uorescence upon addition of Ca2‡ to the solution

685

Ca2‡/Mg2‡-switch in S100P

containing S100P protein are very large (Gribenko et al., 1998). From this we concluded that the addition of Ca2‡ leads to structural rearrangements in the S100P molecule that expose hydrophobic surfaces. This conclusion was based on a common long standing belief that an increase in ANS ¯uorescence is associated with the ANS binding to newly exposed hydrophobic surfaces of proteins (e.g. see Goto et al., 1979; Lakowitcz, 1983; Teschke et al., 1993; Jones et al., 1994). Recently, however, an additional explanation for the changes in ANS ¯uorescence upon interactions with proteins has been suggested. Matulis & Lovrien (1998) have shown that electrostatic interactions between ANS and positively charged groups on proteins can also induce an increase in the ¯uorescence of ANS at neutral pH. Although the details of ANS-protein interactions remain unclear, we have used ANS ¯uorescence to provide an external probe for changes in the S100P structure upon Ca2‡-binding. Figure 6 shows changes in the ¯uorescence of the external probe ANS for WT, Y18F, Y88F and C85S proteins. For all four proteins, the changes in the ANS ¯uorescence are biphasic indicating that both low and high af®nity Ca2‡-binding sites are present in these proteins. This result is particularly important for the Y18F-S100P, for which no apparent biphasic changes were observed in the near-UV CD or Tyr ¯uorescence. Two transitions in Ca2‡induced changes in ANS ¯uorescence indicate that the Y18F substitution did not affect the ability of this protein to bind Ca2‡. Analysis of the Ca2‡binding for all four proteins in terms of a two independent binding sites model (equation (6)) leads to

estimates of the apparent equilibrium binding constants for Ca2‡ on the order of 107 Mÿ1 and 104 Mÿ1, which is similar to those estimated from the near-UV CD and Tyr ¯uorescence data (Table 1). From the results obtained by all three spectroscopic methods, we can conclude that there is a high af®nity and a low af®nity Ca2‡-binding site in the S100P molecule. These sites are located in the C and N-terminal parts of S100P, respectively. Taking into account the cellular concentration of Ca2‡ and the binding af®nities to these two sites, probably only the binding of Ca2‡ to the high af®nity site has functional relevance. Effects of Mg2‡ on the spectroscopic properties of S100P Effects of Mg2‡ on the Ca2‡-binding properties of S100P (WT, Y18F, Y88F, and C85S) have been measured at three different concentrations of Mg2‡; 5 mM, 50 mM and 100 mM. Binding pro®les were monitored using Tyr ¯uorescence (Figure 5) and ANS ¯uorescence (Figure 6). The overall shape of the Ca2‡-binding pro®les for a given protein is independent of the Mg2‡ concentration. However, the analysis of the Ca2‡-binding isotherms in the presence of Mg2‡ reveal an interesting trend: the average apparent Ca2‡-binding constant for the high af®nity site (hK1i) seems to depend on Mg2‡ concentration, whereas the average apparent Ca2‡binding constant for the low af®nity site (hK2i) is independent of Mg2‡ concentration (Table 1). In the absence of Mg2‡ ions hK1i for Ca2‡-binding is

Figure 6. Changes in the ANS ¯uorescence intensity as a function of concentration of Ca2‡ in buffer A for WT-S100P, C85S-S100P, Y18F-S100P and Y88F-S100P. Filled symbols are experimental results obtained at 0 mM (black), 5 mM (red), 50 mM (blue) and 100 mM (green) concentrations of Mg2‡. Lines represent the ®t of the experimental data to equations (5) or (6) using parameters listed in Table 1.

686 8.5
106 Mÿ1 (an averaged value from the analysis of Tyr and ANS ¯uorescence titration pro®les for all four proteins). In the presence of 5 mM Mg2‡, the value for hK1i increases about eightfold to 7.0  107 Mÿ1. Higher, 50 mM or 100 mM, concentrations of Mg2‡ decreases the apparent Ca2‡-binding constant hK1i back to the value observed in the absence of Mg2‡ (8.1  106 Mÿ1 and 4.6  106 Mÿ1 in the presence of 50 mM or 100 mM Mg2‡, respectively). Interestingly, the effect of Mg2‡ on the apparent Ca2‡-binding constant (hK2i) for the low af®nity site is not signi®cant, 1.0  104 Mÿ1, 8.3  103 Mÿ1, 9.4  103 Mÿ1, and 5.4  103 Mÿ1 in the presence of 0, 5, 50 or 100 mM Mg2‡, respectively. Figure 7 presents the changes in ANS ¯uorescence for WT-S100P upon titration with Mg2‡ in the absence and in the presence of Ca2‡. In the absence of Ca2‡, Mg2‡ at concentrations between 10ÿ8 to 10ÿ3 M has little effect on ANS ¯uorescence. Concentrations of Mg2‡ greater than 10ÿ3 M lead to an increase in ANS ¯uorescence. This increase continues up to 100 mM Mg2‡ (pMg ˆ 1) where ANS ¯uorescence reaches a plateau. Increase of Mg2‡ concentration above 100 mM induces an additional increase in ANS ¯uorescence. Analysis of the Mg2‡ titration curve in the absence of Ca2‡ according to the two independent binding sites model (equation (6)) gives an estimate for the apparent binding constants of 5  102 Mÿ1 and 16 Mÿ1. This indicates that Mg2‡ in the absence of Ca2‡ binds to at least two sites to the S100P molecule with one of the apparent binding constants within the physiological range of Mg2‡ concentration. The question is whether Mg2‡ competes with Ca2‡ for the same binding sites or if Mg2‡ binds to different sites? To check these possibilities we performed Mg2‡ titrations of S100P in

Ca2‡/Mg2‡-switch in S100P

the presence of Ca2‡. Titration with Mg2‡ in the presence of 10 mM Ca2‡ (concentration at which the high af®nity C-terminal Ca2‡-binding site is fully saturated) is compared with the titration curve in the absence of Ca2‡ in Figure 7. Initial ANS ¯uorescence (no Mg2‡) in the presence of 10 mM of Ca2‡ is higher than in the absence of Ca2‡ because Ca2‡ increases ANS ¯uorescence (see Figure 6). Addition of Mg2‡ up to 1 mM (pMg ˆ 3) in the presence of 10 mM concentration of Ca2‡ does not change the ANS ¯uorescence. Further increases in the concentration of Mg2‡ leads to an increase in ANS ¯uorescence, which reaches a maximum value at 30 mM Mg2‡ (pMg ˆ 1.5). This is followed by a decrease in ANS ¯uorescence to the original level at higher Mg2‡ concentrations. This decrease explains the effects of 50 and 100 mM Mg2‡ on Ca2‡ titration curves (Figures 5 and 6), i.e. observed lower apparent binding constant of Ca2‡ to the high af®nity binding site both in the absence and in the presence of 50 or 100 mM Mg2‡ than in the presence of 5 mM Mg2‡. It appears that high (50 and 100 mM) concentrations of Mg2‡ abolish the speci®c effect of 5 mM Mg2‡ on Ca2‡-binding. Titration with Mg2‡ in the presence of saturating concentrations of Ca2‡ followed by ANS ¯uorescence is shown in Figure 7. Again, the initial value of ANS ¯uorescence at this concentration of Ca2‡ is much higher than that in the presence of 10 mM Ca2‡ or in its absence. This is associated with the increase in ANS ¯uorescence upon addition of Ca2‡ (Figure 6). Addition of Mg2‡ up to 300 mM to the S100P-ANS solution containing 5 mM Ca2‡ induced only gradual changes in the ANS ¯uorescence. Notably, the values of ANS ¯uorescence at high (>300 mM) concentrations of Mg2‡ under all three conditions

Figure 7. Changes in the ANS ¯uorescence intensity of the WT-S100P as a function of concentration of Mg2‡ (A) and Zn2‡ (B) in 25 mM Tris-HCl (pH 7.5), 0.2 mM EDTA, 3 mM DTT (buffer A). Symbols are experimental results obtained in the absence of divalent ions (*), in the presence of 10 mM Ca2‡ (&), 5 mM Ca2‡ (~), or 5 mM Mg2‡ (!). Continuous lines drawn through the points do not carry any meaning but to guide the eye.

Ca2‡/Mg2‡-switch in S100P

seem to converge, which probably is related to the electrostatic (ionic strength) effects or non-speci®c binding of Mg2‡. The effect of Mg2‡ on the ANS ¯uorescence in the presence of different concentrations of Ca2‡ allows us to propose a possible ligation state of S100P under physiological conditions (micromolar concentrations of Ca2‡ and millimolar concentrations of Mg2‡). The C-terminal (high af®nity for Ca2‡) binding site of S100P is occupied by Ca2‡, whereas the N-terminal (low af®nity for Ca2‡) binding site is occupied by Mg2‡. This conclusion is based on the following experimental observations. First, in the presence of Mg2‡ we see either an increase in af®nity of Ca2‡ to the C-terminal site or no effect at all (Figures 5 and 6). This means that the C-terminal binding site does not bind Mg2‡. Second, the high af®nity binding site for Mg2‡ can be observed in the absence or in the presence of low concentrations of Ca2‡ but not in the presence of saturating concentrations of Ca2‡. This means that when the weak N-terminal Ca2‡-binding site is unoccupied by Ca2‡, Mg2‡ can bind to this site. At saturating Ca2‡ concentrations the weak N-terminal Ca2‡-binding site has Ca2‡ bound and it requires relatively high concentrations of Mg2‡ to replace Ca2‡ at this site. An important aspect of this competition between Mg2‡ and Ca2‡ for the N-terminal binding site is that the apparent binding constants for these two ions at the N-terminal site are different by a couple of orders of magnitude (Table 1). However, under physiological concentrations of these two ions the N-terminal site will be preferentially occupied by Mg2‡, simply because its physiological concentration is at least two orders of magnitude higher. Effects of Zn2‡ on the spectroscopic properties of S100P As opposed to Mg2‡, Zn2‡ appears to bind to the C-terminal high (for Ca2‡) af®nity site. Figure 7B presents the changes in ANS ¯uorescence upon titration with Zn2‡ under four different conditions: in the absence of Mg2‡ or Ca2‡, and in the presence of 10 mM Ca2‡, 5 mM Ca2‡, or 5 mM Mg2‡. In the absence of Mg2‡/Ca2‡ or in the presence of 5 mM Mg2‡, ANS ¯uorescence does not change until the concentration of Zn2‡ reaches 0.1 mM (pZn ˆ 4). Further increase in the Zn2‡ concentration leads to a dramatic increase in ANS ¯uorescence, indicating possible conformational changes in the S100P molecule. Interestingly, changes in the ANS ¯uorescence occur simultaneously in the absence of Mg2‡/Ca2‡ or in the presence of 5 mM Mg2‡, indicating that Zn2‡ does not compete with Mg2‡. This can be considered as an indication that Zn2‡ does not bind to the N-terminal site. When both N and C-terminal sites of S100P are occupied by Ca2‡ (i.e. in the presence of 5 mM Ca2‡), addition of up to 10 mM of Zn2‡ does not induce any changes in the ANS ¯uorescence. This suggests that the C-terminal site is

687 a possible Zn2‡-binding site. Further evidence for this follows from the changes in ANS ¯uorescence upon addition of Zn2‡ in the presence of 10 mM Ca2‡. Under these conditions, the C-terminal site is occupied by Ca2‡, whereas the N-terminal site is empty. The increase in ANS ¯uorescence in this case is shifted (as compared to the Zn2‡ titration curves in the presence of 5 mM Mg2‡or in the absence of both Ca2‡ and Mg2‡) towards higher concentrations of Zn2‡, indicating competition between Ca2‡ and Zn2‡ for the C-terminal binding site. Nevertheless, the concentration range of Zn2‡ where the changes in ANS ¯uorescence are observed is much higher than the physiological concentrations of this metal. Zn2‡ appears to be the most abundant in the brain, but even in this tissue its concentration is only on the order of 10 to 100 nM (e.g. see Ebadi et al., 1981; Fjerdingstad et al., 1974). Thus, a possible physiological regulatory role of Zn2‡ in the functioning of S100P can be ruled out. Analytical ultracentrifugation shows that the apparent molecular mass of S100P changes in response to the presence of Ca2‡ Most of the members of the S100 family of proteins exist in solution as dimers (Hilt & Kligman, 1991; Zimmer et al., 1995; Schafer & Heizman, 1996). The exception is calbindin D9 K, which is a monomer (Skelton et al., 1990a,b, 1995). Figure 8 presents the results of equilibrium centrifugation of WT, Y18F, Y88F and C85S S100P proteins. In the absence of Ca2‡, the effective apparent molecular mass for all proteins is about 20 kDa, a mass that corresponds to the dimer form. Notable curvature of the plot, however, indicates the distribution of populations of different oligomerization states of the S100P molecules. Monomers, dimers and probably trimers and tetramers are possibly present at equilibrium in the absence of Ca2‡. Addition of Ca2‡ leads to different effects on the effective apparent molecular mass, AMWeff, of the S100P proteins (Figure 8). For the WT and Y18F proteins the effective apparent molecular mass changes dramatically. Addition of 5 mM of Ca2‡ increases the AMWeff for these proteins from 20 kDa to 30 kDa (expected molecular mass for the S100P monomer is 10 kDa). We note, however, that the curvature of the plot persists in this case as well. This indicates the presence of different oligomerization states in the WT and Y18F S100P proteins ranging from monomer to possibly tetramer. The effects of saturated concentrations of Ca2‡ on the AMWeff for the Y88F is less than for the WT or Y18F proteins (Figure 8). In contrast, Ca2‡ did not have any signi®cant effect on the AMWeff of the C85S protein. For this protein, the effective apparent molecular mass even decreases from 20 kDa to 17 kDa upon addition of Ca2‡. Effects of Mg2‡ on the effective apparent molecular mass of S100P are different from those of Ca2‡. Figure 8 presents the results of equilibrium

688

Ca2‡/Mg2‡-switch in S100P

Figure 8. Results of analytical centrifugation of S100P proteins in the absence (blue circles) and in the presence (red squares) of 5 mM Ca2‡ or in the presence of 5 mM Mg2‡ (black triangles). Green lines with Roman numerals show the expected plot for monomers (I), dimers (II), trimers (III), and tetramers (IV).

centrifugation of WT-S100P in the presence of different concentrations of Mg2‡. Addition of this divalent cation does not produce changes in the effective apparent molecular mass of the S100P protein. This shows that the changes in the monomer-oligomer equilibrium are perturbed by Ca2‡ but not Mg2‡. Keeping in mind that Mg2‡ probably does not bind to the high af®nity C-terminal site, we propose that the changes in the oligomerization state occur only when both C and N-terminal sites are ligated. The results of equilibrium centrifugation indicate that in the absence of Ca2‡, S100P exists in solution as an equilibrium mixture of monomers and oligomers (dimers, trimers and tetramers). Addition of Ca2‡ perturbs this equilibrium towards the higher order of oligomers. The fact that C85S, and to a lesser degree Y88F, respond differently to the addition of Ca2‡ provides an indication that these residues are possibly involved in the formation of the oligomerization interface, forming dimers, trimers and/or tetramers. The question about the relative population of each species in the solution remains open, particularly because the analytical ultracentrifugation experiments are performed at relatively high concentrations (1 to 9 mg/ml) due to the low extinction coef®cient of S100P. Gel filtration profiles of S100P show dependence on concentration of Ca2‡and Mg2‡ Figure 9 shows the elution volumes of the S100P proteins as a function of Ca2‡ concentration. In the

absence of Ca2‡ all proteins have an elution volume of 11.5 ml, which is practically independent of the protein concentration. In the presence of 5 mM Ca2‡ the elution volumes of the S100P proteins increase from 11.5 ml to 12.8 ml (Figure 9). This increase in the elution volume can be explained by a decrease in the hydrodynamic size of S100P due to conformational changes in the molecule and a decrease in the ¯exibility of the Ca2‡-binding loops upon ligand binding. The increase in the elution volume of S100P upon addition of 5 mM Ca2‡ is in apparent contradiction with the results of equilibrium centrifugation, which show an increase in the apparent molecular mass. However, one needs to keep in mind two important differences between gel-®ltration chromatography and analytical centrifugation. First, analytical centrifugation is an equilibrium experiment, while gel-®ltration experiments depend on the time regime of the observed process as compared to the separation time (e.g. see Hilser & Freire, 1995). Second, the elution volume of the molecules in gel-®ltration chromatography depends on both the size (molecular mass) and the shape of the macromolecule. In the case of S100P, Ca2‡-binding leads to notable conformational changes (Figures 3 to 6). Upon binding of Ca2‡, the ¯exibility of the Ca2‡-binding loops will decrease, which will in turn decrease the Stokes radius and increase the elution volume of S100P. Another evidence that a decrease in Stokes radius is due to a Ca2‡-dependent decrease in the ¯exibility of the

Ca2‡/Mg2‡-switch in S100P

689 Ca2‡, i.e. no changes in elution volume are observed. The addition of 10 mM Ca2‡ to the S100P solution containing 5 mM Mg2‡ leads to an increase of the elution volume to 12.5 ml, i.e. to the value observed in the presence of only 5 mM Ca2‡. Since Mg2‡ binds only to the N-terminal binding site on the S100P molecule, the observed changes in the elution volume occur only when both C-terminal and N-terminal sites are ligated. Effects of Ca2‡ and Mg2‡ on the conformation of S100P in solution

Figure 9. The dependence of the elution volume of S100P on the concentration of Ca2‡ in elution buffer B for WT-S100P (*), C85S-S100P (}), Y18F-S100P (~), and Y88F-S100P (&). Filled circles (*) show the elution volume of WT-S100P in the presence of 5 mM Mg2‡. The continuous line is the linear ®t of all data points obtained in the absence of Mg2‡ to the equation (5), using K1 ˆ 2.3  104 Mÿ1. Dotted lines show the elution volumes for ovalbumin (43 kDa), interleukin-1b (18 kDa), and RNase T1 (12 kDa).

S100P molecule follows from the analysis of the dependence of the elution volume on the concentration of Ca2‡ in the elution buffer (Figure 9). An increase in the elution volume from 11.5 ml to 12.8 ml with the increase of Ca2‡ concentration is not monotonous but seem to represent a titration curve. Analysis of these data in terms of a single site Ca2‡-binding model (equation (3)) gives an estimate of the apparent binding constant of 2.3  104 Mÿ1. This estimate for the apparent Ca2‡ binding constant is in good correspondence with the apparent binding constants estimated using spectroscopic techniques for the weak Ca2‡-binding site (Table 1). Somewhat smaller changes in the Stokes radius have been observed upon Ca2‡-binding to S100B (Mani & Kay, 1984; Drohat et al., 1997). Changes in the shape of the S100A protein upon Ca2‡-binding have also been demonstrated (Wang et al., 1992). Thus, binding of the Ca2‡ to the weak N-terminal binding site is causing the change in the elution volume of the S100P protein. If binding to the N-terminal site triggers changes in the size/shape of the S100P molecules, binding of Mg2‡ to this site should also have a similar effect. This is indeed the case (Figure 9). However, in order for Mg2‡ to produce such a change in the elution volume, the C-terminal site must be saturated with Ca2‡. The elution volume of WT-S100P in the absence of Ca2‡ but in the presence of 5 mM Mg2‡ is 11.5 ml, similar to that in the absence of

The S100P molecule contains two ion binding sites, one located in the N-terminal part of the molecule and the second in the C-terminal part. The C-terminal binding site has high af®nity to Ca2‡ and does not bind Mg2‡. The N-terminal site binds both Ca2‡ and Mg2‡. In the absence of Ca2‡, S100P adopts a conformation in which both tyrosine residues (18 and 88) appear to be solvent exposed and shows no speci®c near-UV CD signal in the 275 to 290 nm range (Figure 3). Under these conditions, the protein exists in an equilibrium between monomeric and oligomeric species. Oligomeric species consist, even at high protein concentrations, primarily of dimers as judged by the equilibrium centrifugation experiments (Figure 8). In the presence of low (up to 10 mM) Ca2‡ concentrations, the C-terminal binding site binds Ca2‡. The apparent binding constant for this high af®nity site in the absence of other divalent ions is on the order of 107 Mÿ1 (Table 1). Binding of Ca2‡ to this site induces conformational changes in the S100P molecule. These conformational changes are propagated throughout the molecule leading to the structural rearrangements involving both tyrosine residues, as judged by the appearance of the positive ellipticity in near-UV CD spectra, and a decrease in the Tyr ¯uorescence intensity (Figures 3 to 5). The only cysteine residue at position 85 becomes more accessible for chemical modi®cation by DTNB, indicating greater exposure of this residue to the solvent (data not shown). Conformational changes also involve possible exposure of large hydrophobic surfaces, if the increase in ANS ¯uorescence intensity can be interpreted in these terms (Figure 6). The equilibrium between different oligomeric species remains similar to that in the absence of Ca2‡. It appears that the Ca2‡ binding af®nities to different oligomeric species are very close because experiments performed at different protein concentrations (14 mM in ¯uorescence experiments and 400 mM in near-UV CD experiments) give comparable apparent equilibrium constant (Table 1). At concentrations of Ca2‡ above 10 mM, Ca2‡ binds to the N-terminal binding site. The apparent binding constant for this low af®nity site is on the order of 104 Mÿ1 (Table 1). Conformational changes upon binding to the N-terminal binding site again involve structural rearrangements in the structure

690 of S100P. Both near-UV CD spectroscopy and Tyr ¯uorescence intensity show changes comparable in magnitude to those observed upon Ca2‡-binding to the C-terminal site. However, the changes in the ANS ¯uorescence for the binding of the second Ca2‡ are much smaller in amplitude than those for the ®rst Ca2‡ (Figure 6 and Table 1). The equilibrium between different oligomeric species is shifted towards higher population of oligomeric species, leading to an effective apparent molecular mass of 30 kDa (Figure 8). The hydrodynamic properties of the oligomeric species also change: they elute off the gel-®ltration column signi®cantly later, indicating a decrease in the hydrodynamic size of the species. This decrease in the hydrodynamic size is probably associated with a decrease in the ¯exibility of the Ca2‡-binding loops and some additional structural rearrangements. Mg2‡ also interacts with the S100P molecule. It appears that Mg2‡ does not bind to the C-terminal binding site but only to the N-terminal site. The af®nity of the N-terminal site to Mg2‡ is only an order of magnitude lower than the af®nity of Ca2‡, 102 Mÿ1 for Mg2‡ and 103 Mÿ1 for Ca2‡ (Table 1). Binding of Mg2‡ to this site seems to enhance the Ca2‡ binding to the C-terminal binding site by about eight fold, indicating positive cooperativity between these sites. Comparison of S100P with calbindin D9k, S100B and calcyclin Conformational properties observed for S100P can be compared with the three other well studied proteins of S100 family: calbindin D9k, S100B and calcyclin (S100A6). Calbindin D9k, in contrast to all other S100 proteins, is a monomeric protein of 78 amino acid residues. It binds two divalent ions per protein molecule (Svensson et al., 1992). Binding constants for Ca2‡ for these binding sites differ by an order of magnitude and range from 108/109 Mÿ1 in low salt to 106/107 Mÿ1 in 150 mM salt (Linse et al., 1987; Akke et al., 1993). These binding constants are signi®cantly higher than those for S100P. However, in contrast to S100P, calbindin D9k can bind Mg2‡ at both sites with af®nities which differ by two orders of magnitude: 105/103 Mÿ1 in low salt to 103/101 Mÿ1 in 150 mM salt (Andersson et al., 1997). Binding of Mg2‡ decreases the af®nity of Ca2‡ to the calbindin D9k molecule (negative cooperativity; Andersson et al., 1997), which is just opposite to what is observed for S100P. Both ions (Ca2‡ and Mg2‡) induce signi®cant structural changes in the calbindin D9k molecule, as observed by X-ray and NMR spectroscopies (Skelton et al., 1990a,b, 1995). S100B is a typical protein of the S100 family. Analytical ultracentrifugation and dynamic light scattering experiments on rat S100B showed that both in the presence and in the absence of Ca2‡, the S100B exists in solution as a dimer (Landar et al., 1997). This result has been further supported

Ca2‡/Mg2‡-switch in S100P

using gel-®ltration chromatography (Drohat et al., 1997). Thus, the oligomerization and hydrodynamic properties of the S100P molecule differ signi®cantly from those for S100B. Furthermore, substitution of Cys84 to Ser did not change the oligomerization state of the S100B protein neither in the absence nor in the presence of Ca2‡ (Landar et al., 1997). The Cys84 in the S100B sequence is equivalent to the Cys85 in the S100P sequence, for which C85S substitution abolishes the effect of Ca2‡ on the apparent molecular mass as determined by analytical ultracentrifugation (Figure 8). The S100B dimer binds four Ca ions with a relatively low, as compared to S100P, af®nity (105 Mÿ1) (Mani et al., 1983; Baudier & Cole, 1989). In contrast to S100P or calbindin D9k, S100B does not bind Mg2‡ (Baudier & Gerard, 1983). However, it has been shown that another divalent metal, Zn2‡, can bind to S100B with the stoichiometry of at least four Zn2‡ molecules per S100B dimer (Baudier et al., 1986; Baudier & Cole, 1989; Mani & Kay, 1987). The af®nity of S100B for Zn2‡ is at least two orders of magnitude higher than for Ca2‡. This high af®nity Zn2‡-binding is in contrast with the effects of Zn2‡ on S100P. The S100A6 (calcyclin) protein, another member of S100 family, also exists as a dimer in solution and binds four Ca2‡ molecules per protein dimer (Potts et al., 1996; Sastry et al., 1998). Although both S100B and S100A6 bind Ca2‡ with similar stoichiometry and af®nity, the conformational changes which accompany Ca2‡ binding are dramatically different. Solution structures of S100A6 in the presence (Sastry et al., 1998) and absence (Potts et al., 1996) of Ca2‡ show that there are only modest changes in the conformation of the protein upon Ca2‡ binding. In contrast, S100B shows dramatic changes in the conformation (Drohat et al., 1998; Matsumura et al., 1998; Smith & Shaw, 1998). The major changes involve the relative orientation of the helices in the C-terminal EF-hand. Upon binding of Ca2‡, helix III changes its orientation relative to helices I, II and IV. These conformational changes involve exposure of hydrophobic surfaces (Drohat et al., 1998; Matsumura et al., 1998; Smith & Shaw, 1998). From the low resolution structural information (near-UV CD, Tyr ¯uorescence and ANS ¯uorescence) we have for S100P it is reasonable to suggest that Ca2‡ binding to S100P induces structural changes, probably similar to that of S100B, i.e. exposure of hydrophobic surfaces involving helix III. This conclusion follows not only from the increase in ANS ¯uorescence of S100P upon Ca2‡ binding, but also from the increased accessibility of Cys85 to DTNB (data not shown). Overall, S100P shares certain conformational properties with all three proteins, calbindin D9k, S100B and calcyclin, but nevertheless a combination of these properties is novel for the S100 proteins.

691

Ca2‡/Mg2‡-switch in S100P

Model for functional activity of S100P as Ca2‡/Mg2‡-switch The results presented above provide a simpli®ed model for the functional activity of S100P as a Ca2‡/Mg2‡-switch under physiological conditions (pH 7.5, concentration of Ca2‡ under 10 mM and concentration of Mg2‡ 5 mM). Depending on the concentrations of Ca2‡ and Mg2‡ S100P can exist in four different conformational states: apo-state, CCa2‡-state, NMg2‡-state, and CCa2‡NMg2‡-state (Figure 10). The apo-state is populated at low concentrations of both cations, Ca2‡ and Mg2‡. Increase in concentration of Ca2‡ when Mg2‡ is still scarce produces the CCa2‡-state of the protein in which the C-terminal site is ligated by Ca2‡. Conversely, increase in the Mg2‡ concentrations when Ca2‡ levels are low, will produce the NMg2‡-state of S100P. In this state the Nterminal binding site is occupied by Mg2‡. Consequent increase in Ca2‡ concentrations will lead to the formation of the CCa2‡NMg2‡-state. This state can also be populated from the CCa2‡-state by an increase in Mg2‡ concentration. Binding of Ca2‡ to the C-terminal site is enhanced by the presence of the Mg2‡ bound to the N-terminal site, i.e. af®nity of Ca2‡ to the apo-state appears to be lower than to the NMg2‡-state. This model describes possible general properties of the S100P protein as a Ca2‡/Mg2‡-switch. It does not incorporate, however, any detailed effects of the Ca2‡ and/or Mg2‡ on the oligomerization state of S100P. More studies will be required to provide suf®cient experimental data on the oligomerization state of S100P in solution and detailed structural properties of the NMg2‡ and the CCa2‡states.

Materials and Methods Construction and purification of S100P mutants Cloning and puri®cation of recombinant wild-type S100P have been reported (Gribenko et al., 1998). The Y18F, Y88F, and C85S mutants were constructed by sequential PCR step mutagenesis (Cormack, 1992) using the primers: Y18F-forward, 50 -TTTTCCCGATTTTCGGGCAGG; Y18F-reverse, 50 -GGTGCCGGAAAATCGGGAAAA; Y88F-forward, 50 -TGTCACAAGTTCTTTGAGAAG; Y88F-reverse, 50 -CTTCTCAAAGAACTTGTGACA; C85Sforward, 50 -ACGTCTGCCTCTCACAAGTAC; C85Sreverse, 50 -GTACTTGTGAGAGGCAGACGT. The mutations were identi®ed by sequencing on an ABI Prism 310 genetic analyzer using a T7 primer. All variants have been puri®ed to homogeneity according to the procedure described for the wild-type protein (Gribenko et al., 1998). Sample preparation Lyophilized proteins were redissolved in 20 mM TrisHCl (pH 7.5), 1 mM EDTA and incubated with 2% (v/v) b-mercaptoethanol at room temperature for a minimum of two hours. The protein solution was further dialyzed against the corresponding buffer in Spectrapor 3 membranes (molecular mass cutoff of 3500 Da) for at least 15 hours using several changes of buffer. To reduce Ca2‡ contamination, dialysis and all other procedures were performed in plastic containers. For spectroscopic experiments all buffers contained buffer A (25 mM Tris-HCl (pH 7.5), 0.2 mM EDTA, 3 mM DTT) in addition to various concentrations of CaCl2, MgCl2 or ZnCl2. For gel ®ltration chromatography and analytical centrifugation all buffers contained buffer B (50 mM Tris (pH 7.5), 1 mM DTT, 0.2 mM EDTA). Protein concentration was determined spectrophotometrically using an extinction coef®cient E0.1% 1 cm,280 nm of 0.292 for S100P wild-type and C85S-S100P, 0.146 for Y18F-S100P and Y88F-S100P, calculated according to Gill & von Hippel (1989) as described by Pace et al. (1995). Light scattering correction was taken into account according to Winder & Gent (1971). Calcium solutions used in the experiments were standardized using Perkin-Elmer AA 2380 atomic absorption spectrometer. Serial dilutions of calcium atomic absorption standard at 1000 mg/ml (Aldrich) were used to calibrate the instrument. Fluorescence spectroscopy

Figure 10. A ``Ca2‡/Mg2‡-switch'' model for the effects of Ca2‡ and Mg2‡ on the conformational properties of S100P in solution.

Steady-state ¯uorescence experiments were performed on FluoroMax Spectro¯uorimeter with DM3000F software (SPEX Industries, Inc.). Constant temperature of the solution (25 C) was maintained using a circulating water bath (Fisher Scienti®c). A quartz cell with 1 cm path length was used. All experiments were run in triplicate and the average value reported. For tyrosine ¯uorescence Ca2‡ titration experiments the excitation wavelength used was 276 nm, and the emission wavelength was 306 nm. The protein concentration was 0.15 mg/ml for S100P wild-type and S100PC85S, 0.3 mg/ml for S100P-Y18F and S100P-Y88F. Samples were titrated with increasing amounts of calcium to saturation. The average of 100 readings was recorded at each titration point. Intensity at each point was corrected for dilution.

692

Ca2‡/Mg2‡-switch in S100P

For 8-anilino-1-naphtalenesulfonic acid (ANS) ¯uorescence experiments, ANS was added to the buffer to a ®nal concentration of 30 mM. The excitation and emission wavelengths used were 350 nm and 515 nm, respectively. The protein concentration in all experiments was 0.3 mg/ml. The concentration of stock solution of ANS was determined spectrophotometrically using an extinction coef®cient of 5000 Mÿ1 cmÿ1 at 355 nm.

Circular dichroism spectroscopy Ellipticity measurements were performed on a JASCO J-20 automatic recording spectropolarimeter equipped with a photoelastic modulator and a lock-in ampli®er. The signal from the lock-in ampli®er was passed through a microvolt ammeter (Kethley/Metrabyte) and collected on a PC computer via a 12 bit A/D board. The electrical signal was calibrated in ellipticity units using known values for ellipticity of aqueous solution of d-(‡)-10-camphorsulphonic acid (Yang et al., 1986). Far-UV CD spectra were measured in 1 mm quartz cell. The concentration of all protein samples was 0.15 mg/ml. Quartz cells of 10 mm optical path length were used for the near-UV CD experiments. Three independent experiments with protein concentrations ranging from 3 to 6 mg/ml were performed for each data point. The titration curves were obtained by adding increasing amounts of ions to the protein solution until reaching saturation. One-hundred ellipticity readings at each wavelength (276, 268, and 262 nm) were averaged for every titration point. Ellipticity values were corrected for dilution. Molar ellipticity [] was calculated as: ‰Š ˆ

100    MW Cl

…1†

where  is the experimental ellipticity, MW is the mean molecular mass of the amino acid residues in S100P, C is the concentration of protein in solution, and l is the optical path length.

Gel-®ltration experiments were performed on a Superose 12 HR 10/30 (Pharmacia-LKB Biotechnology) column at 25 C at a ¯ow rate of 0.5 ml/min. The elution pro®le was monitored with a UV detector at 280 nm using either FPLC (Pharmacia) or BioCad (Perseptive Biosystems) systems. The column was calibrated using ovalbumin (43 kDa), interleukin 1b (18 kDa) and RNase T1 (12 kDa). Analysis of binding isotherms Changes observed in a spectroscopic signal (Tyr ¯uorescence, ANS ¯uorescence, or near-UV CD spectroscopy), Qexp, upon addition of Ca2‡ were assumed to be proportional to the degree of Ca2‡-binding Ca2‡, X. The degree of binding was expressed either as a single site binding equation: Xˆ

Analytical ultracentrifugation experiments were performed on the Beckman XLA centrifuge. The pro®les were scanned at 276 nm. All experiments were performed at 22,000 rpm and 25 C. At least three different protein concentrations with optical densities at 276 nm of 0.4, 1.0, and 1.4 o.u in a 1 cm cell were used for each WT, Y18F, Y88F, and C85S S100P proteins. Two sets of solvent conditions were used: buffer A and buffer A plus 5 mM CaCl2. The apparent weight average molecular mass, Mw,app, was calculated from the following equation: …2†

where ln(Ar) is the natural logarithm of the absorbance reading at the radial distance r, r is the density of the solution taken to be equal to the 1 g/cm3, o is the rotor angular velocity, and n is the partial speci®c volume of S100P in aqueous solution. The partial speci®c volume of S100P was estimated from its amino acid composition, according to Makhatadze et al. (1990) and was found to be 0.749 cm3 gÿ1 at 25 C.

K1  ‰Ca2‡ Š 1 ‡ K1  ‰Ca2‡ Š

…3†

or as a binding to two independent binding sites: X ˆ X1 ‡ X2 ˆ

K1  ‰Ca2‡ Š K2  ‰Ca2‡ Š ‡ 1 ‡ K1  ‰Ca2‡ Š 1 ‡ K2  ‰Ca2‡ Š

…4†

where [Ca2‡] is the total concentration of Ca2‡; X1 and X2 are the degree of binding for the ®st and second binding sites, respectively; K1 and K2 are apparent binding constants for ®rst and second binding sites, respectively (Wyman & Gill, 1990). The experimental signal, Qexp, is thus related to the degree of binding for a single site binding model: Qexp ˆ Qo ‡ Q1 X ˆ Qo ‡ Q1

K1  ‰Ca2‡ Š 1 ‡ K1  ‰Ca2‡ Š

…5†

or for a two independent binding site model: Qexp ˆ Qo ‡ Q1 X1 ‡ Q2 X2 ˆ Qo ‡ Q1 ‡ Q2

Analytical equilibrium ultracentrifugation

2RT  ln…Ar † ˆ Mw;app  r2 …1 ÿ r  n †  o2

Gel-filtration chromatography

K2  ‰Ca2‡ Š 1 ‡ K2  ‰Ca2‡ Š

K1  ‰Ca2‡ Š 1 ‡ K1  ‰Ca2‡ Š …6†

where Qo is the spectroscopic signal for apo-protein, and Q1 and Q2 are the maximal changes in the spectroscopic probe induced by the binding events. Experimental data were ®tted to equation (5) or (6) using non-linear regression software (NLREG) supplied by Phylip Sherrod.

Acknowledgements We thank Dr Maria M. Lopez for the help with the ¯uorescence experiments; Susan Thomas for helpful discussions; Professor Katsuhide Yutani for use of the Beckman XLA centrifuge; Dr Dennis Shelly for the use of the atomic absorption spectrometer; and Drs Robert Fox, James Lee and James Harman for useful advise. This work was supported in part by the Research Innovation Award from Research Corporation (RI0045), by the grant from the South Plains Foundation, and by a Monboshou International collaboration research grant.

Ca2‡/Mg2‡-switch in S100P

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Edited by P. E. Wright (Received 4 June 1998; received in revised form 27 July 1998; accepted 29 July 1998)