Results and discussion

Overall structures of the three Zn²⁺-Ca²⁺-S100B

We have determined the structures of Zn²⁺-Ca²⁺-loaded human-S100B at pH 6.5, 9.0 and 10.0 (Table. 2). The protein crystallized in the presence of equimolar amounts of Zn²⁺

(one Zn²⁺ per subunit) and excess amounts of Ca²⁺. The structures revealed a high-occupancy sites for both Zn²⁺ and Ca²⁺. There are two Zn²⁺ ion and four Ca²⁺ ions bound per dimer. The overall fold of the human Zn²⁺-Ca²⁺-S100B is similar to that of other S100 proteins like the human Zn²⁺-Ca²⁺-S100A7 and the Cu²⁺-Ca²⁺-S100A12 structure (Fig. 5 C). The overall folds of the three presented structures are virtually identical.

Adjacent subunits are stabilized mainly by hydrophobic interaction. The alignments of the main-chain atoms (aa 1-89) of the Zn²⁺-Ca²⁺-S100B structures (pH 6.5 and pH 9.0 aligned against the pH 10.0 structure) exhibit no significant difference within their structures (RMSD from 0.178Å to 0.19Å). Figure 1 A shows the overall fold of one Zn²⁺-Ca²⁺-S100B at pH 10 and is representative for the structures determined at pH 6.5 and pH 9.0. The Ca²⁺ ions are bound in the two EF-hands, whereas the Zn²⁺ ions are bound at interface (Fig. 2 A-C). The structure of Zn²⁺-Ca²⁺-loaded human S100B is very similar to the structures of S100B in complex with Ca²⁺ or Ca²⁺ and peptides derived from target proteins like TRTK-S100B (1MWN), NDR-S100B (1PSB) and p53^367-388 (1DT7) (Bhattacharya et al., 2003; Inman et al., 2002; Rustandi et al., 2000). However, there are some noticeable differences between the X-ray structures of Zn²⁺-Ca²⁺-S100B when compared to other structures of S100B. In a recent study, it was shown that binding of Zn²⁺ to Ca²⁺-loaded S100B leads to the formation of a kink in helixIV, close to their C-terminus and that the dimers rearrange to a denser packing, in the zinc loaded state (Fig. 1 C) (Wilder et al., 2005). The structures of human Zn²⁺-Ca²⁺-S100B presented in this study, revealed neither a kink formation nor a denser packing. Remarkably, the binding of Zn²⁺ to Ca²⁺-S100B results only in a minor rearrangement of the C-terminus, which induces an elongation of helix IV by 5 residues compared to Zn²⁺ free Ca²⁺-S100B (Fig. 1A and 3). To test whether there is a Zn²⁺-dependent conformational change in the α-helical content in Zn²⁺-Ca²⁺-loaded S100B, as observed in the crystal structures, CD spectra of S100B in the Ca²⁺ loaded and Zn²⁺-Ca²⁺-loaded state were recorded and

compared. In general, the CD spectra of Ca²⁺-S100B are very similar to those of Zn²⁺ -Ca²⁺-S100B. Nevertheless, upon addition of Zn²⁺ there was a minor change in the CD spectrum, indicating an increased α-helical content most likely the elongation in helix IV (Fig. 6.). This extension of the α-helix was also observed in the rat Zn²⁺-Ca²⁺-S100B NMR structure by Wilder et. al.

This helix extension causes a reorientation of several residues, which are involved in target recognition. Particular Phe87 and Phe88, which now point towards the target-binding cleft and adopt a conformation that is observed in the Ca²⁺-S100B NDR-Kinase peptide complex. In the structures reported here, the positions of these residues 86-89, especially the hydrophobic Phe87 / 88, are well defined as documented by comparatively low B-factors. The rearrangement of the C-terminal residues Phe87 and Phe88 upon Zn²⁺

binding enlarges the hydrophobic surface of S100B, which is essential for the interaction with the phenylsepharose matrix. Studies of Baudier et, al., showed that S100B could be purified solely by Zn²⁺ depended interaction, with hydrophobic media such as phenylsepharose (Baudier et al., 1982). The binding of Zn²⁺ results in the positioning of some hydrophobic residues that are necessary for ligand binding. Such an arrangement of the hydrophobic residues Phe87 and Phe88 possibly could induce the 5-fold higher affinity of bound TRTK-12 peptide from CapZ to the Zn²⁺-Ca²⁺-S100B than to the Ca²⁺ -S100B protein (Barber et al., 1999). One can assume that ligand binding to a Zn²⁺-loaded Ca²⁺-S100B may be promoted, compared to the Zn²⁺-free Ca²⁺-S100B. Besides this, the binding of Zn²⁺ could also affect the binding affinity of the NDR-Kinase to S100B in a positive manner by stabilizing the binding site at the C-terminus of S100B. The NMR structure of the bovine Ca²⁺-S100B in complex with the N-terminal regulatory domain fragment of the NDR-Kinase showed an interaction of the Phe87 with the peptide fragment (Bhattacharya et al., 2003). This finding indicates that S100B becomes highly hydrophobic upon Zn²⁺ binding. Since Zn²⁺ cannot replace the Ca²⁺ ions in the EF-hands and thereby induce the Ca²⁺ dependent conformational change, the mechanism must be different. Most likely Zn²⁺ alone already promote a Ca²⁺ bound like conformation, in the N-terminal EF-hand loop. A more pronounced effect of Zn²⁺ binding alone to S100B is reflected in the ability of the protein to induce total disassembly of microtubules in the presence of µM Zn²⁺ concentrations were Zn²⁺-S100B leads to an inhibition of τ phosphorylation by protein kinase II (Baudier and Cole, 1988), possibly by Zn²⁺

sequestration. It might be possible that the C-terminus of S100B form a functional Zn²⁺

binding site including the residues Phe87 and Phe88 which can cooperate independent

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from the Ca²⁺ binding EF-hands, therefore S100B may exerts besides a Ca²⁺ dependent function also a Zn²⁺ dependent function, without Ca²⁺. In all these cases, Zn²⁺ binding enables Ca²⁺-S100B to an additional way to modulate the dissociation rate for bound ligands. The interhelical angles (IHAs) between helices I and II and also the IHAs of helices III and IV are important since they participate on the S100-specific- (I-II) and the classical- (III-IV) Ca²⁺ binding EF-hands. A comparison of the interhelical angles within the three Zn²⁺-Ca²⁺-S100B structures indicate that the angles and distances of the flanking helices of the S100-specific EF-hand are virtually identical (I-II = 137.7 ± 0.4°, 15 Å; III-IV = 106,2 ± 0.8°, 15 Å, Table: 4). A difference was detected for the flanking helices III and IV of the classical EF-hand, were the comparison of the human Ca²⁺ -S100B crystal structure (2H61) with the human Zn²⁺-Ca²⁺-S100B crystal structures shows only a minor effect. Here, the addition of Zn²⁺ to the Ca²⁺-S100B induces only a small change to a more open conformation, shown by an increase of the interhelical angles between helices III and IV of 2°-3°. In the case of the Zn²⁺-Ca²⁺-S100B NMR structure from rat compared the Ca²⁺-S100B NMR structure this change was larger, here Wilder et al., found a more opened binding cleft of 12°. on the interhelical angles upon Zn²⁺ binding (Table: 3). Combined, the Zn²⁺ binding does not induce any change in the interhelical angles of both EF-hands but it favors an enlargement of the hydrophobic surface at the C-terminus and therefore may contribute to the binding affinity of Zn²⁺ -Ca²⁺-S100B to its ligands.

The Zn²⁺-binding site

In previous studies, the divalent cation binding properties of the human S100B was studied by Baudier et. al. and it was found that S100B binds four Ca²⁺ and 6-8 Zn²⁺ (two high affinity plus 4-6 low affinity) atoms per dimer. The affinity of S100B for Zn²⁺ is considerably higher than that for Ca²⁺. The binding constant for the Zn²⁺ ion was measured and determined to ~100 nM. (Baudier et al., 1986; Rustandi et al., 1998). The existence of two or more Zn²⁺-binding sites per S100B dimer has been described previously in a number of biochemical studies and a recent structural NMR study (Wilder et al., 2005). The strong binding of the Zn²⁺ to S100B is achieved through four residues of a preformed binding site (Fig.2 A-C). The distance between the two Zn²⁺ ions in the structures is approximately 30.4 Å. Interestingly, alignments of the main-chain

atoms (residues 1-89) of human Ca²⁺-S100B subunits with the obtained Zn²⁺-Ca²⁺ -S100B structures show no significant changes upon binding of Zn²⁺, neither in the overall structure (not shown) nor in the EF-hand Ca²⁺ binding loops (Fig. 5B). Crystal structures obtained at pH 6.5 and pH 10.0 show two histidines His15, His25 from one subunit and one histidine, His85´ and a Glu89´ from the second subunit coordinating Zn²⁺. The Zn²⁺coordination is obtained by the imidazoles of the histidines and the with the terminal carboxylate oxygen atom of the glutamate. The O-Zn and N-Zn distances at the Zn²⁺ binding sites are comparable to other Zn²⁺ binding sites (Table: 4) (Harding, 2000).

Furthermore, the Zn²⁺ binding site ties the two subunits more together, since two of the coordinating ligands of at Zn²⁺ site originate from one subunit and the other ligands are derived from the second subunit (Fig. 2 A-C). Remarkably, in the crystal structure obtained at pH 9.0, the Zn²⁺ ion is coordinated by four histidines (His15, His25, His85´

and His90´). In all three Zn²⁺-Ca²⁺-S100B structures, the Zn²⁺ ligand is coordinated in a slightly distorted tetraeder (Fig. 2A-C). A similar coordination was shown for Cu²⁺ in the Cu²⁺-Ca²⁺-S100A12 structure by three histidines His15, His85´ and His89´ and Asp25 (Moroz et al., 2003) and for the Zn²⁺-Ca²⁺-loaded S100A7 were Zn²⁺ is coordinated by His15, His85´ and His89´ and Asp25 (Fig 4A-C). It is proposed that S100B binds Cu²⁺ at the same site. As depicted in figure four were coordinating residues are superimposed.

For the Zn²⁺ (S100B and S100A7) and the Cu²⁺ (S100A12) loaded structures the positions of the residues are largely the same, with a few exceptions. Sequence alignments showed that only the calgranulins S100A8, S100A9 and S100A12, as well as S100B and S100A7 have such a common Zn²⁺-/Cu²⁺-binding motif which is highly conserved in the homologues proteins in different species (Moroz et al., 2003).

Interestingly, these Zn²⁺-binding S100-proteins have an extracellular function. There they can exert Zn²⁺ dependent function distinct from intracellular functions, but in both circumstances, Zn²⁺-binding promotes ligand-binding affinity of S100-proteins. In general, once formed, Zn²⁺ binding site seems to be very stable in S100-proteins, which were shown by previous, and our recent study (pH range 6.5 to 10.0). Similar pH stable Zn²⁺ sites have been described for the X-ray structures of S100A7 (pH range 6.5 to 8.0) (Brodersen et al., 1999).

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The EF-hand Ca²⁺-binding sites

Binding of Zn²⁺ induces no conformational change in both Ca²⁺ loaded EF-hands, (Fig.5 B-C, data not shown for the classical EF-hand). Although His25 is located at the S100-specific N-terminal EF-hand, its conformation remains essentially the same upon Zn²⁺

binding. The 10-fold increase in Ca²⁺ affinity towards S100B (Baudier et al., 1986) in the presence of Zn²⁺ is explained in that way that Zn²⁺ binding leads to a pre positioning of the N-terminal EF-hand loop which will favor the following Ca²⁺-binding (Fig. 6 A).

One can presume, that the N-terminal EF-hand loop is brought into a virtual Ca²⁺ loaded position and therefore promote the higher binding affinity for Ca²⁺. The r.m.s. deviation between the main-chain atoms (overall) of the human Ca²⁺-S100B structure and the three Zn²⁺-Ca²⁺-S100B structures comes to 0.33 Å ± 0.02 Å. Superposition of the S100-specific EF-hand in the Ca²⁺-S100B structure with the Zn²⁺-Ca²⁺-S100B structure show no particular difference in the EF-hand loop, with the exemption that the coordinating residue His25 rearrange towards the Zn²⁺ (Fig. 6 B). Similarly of the N-terminal EF-hand of Cu²⁺-Ca²⁺-S100A12 with the Zn²⁺-Ca²⁺-S100B shows that in both structures the EF-hand loops were kept in the very same position (Fig. 6C). The same was documented for the EF-hand region in the S100A7 Zn²⁺-loaded structure (Brodersen et al., 1999).

Besides this there was no apparent structural change in the C-terminal classical EF-hand loop. Thus, one can assume that the Ca²⁺-loaded N-terminal EF-hand provides already an ideal topology and geometry for Ca²⁺-binding. It is to assume that the binding of Zn²⁺

stabilizes the structure at the Ca²⁺-loaded N-terminal EF-hand, by inducing a more rigid loop conformation that might mimic a Ca²⁺-loaded like topology, which promotes calcium binding..

ACKNOWLEDGEMENTS

We thank W. Welte for providing the X-ray facilities at the University of Konstanz. This work was partially supported by the grants of the Deutsche Forschungsgemeinschaft (DFG, TR-SFB11), of the SNF (Grant No. 3100A0-1o970) and the Wilhelm Sander-Stiftung. Data of the crystals where collected at the Swiss Light Source (SLS) in Villingen / Switzerland.

3.4.5 Figures

Figure 1: A: Overall structure of the human Zn²⁺-Ca²⁺-S100B dimer at pH 10.0 (light-blue, structures obtained at pH 6.5 and pH 9.0 show the same overall fold). B: Overall structure of human Ca²⁺-S100B structure (red; PDB entry 2H61). C:

Overall fold of the Zn²⁺-Ca²⁺-S100B rat NMR structure (light orange;

PDB entry 1XYD). The dimers are depicted as cartoons and the Ca²⁺

ions are shown as yellow spheres, the Zn²⁺ ions are shown as magenta spheres.

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Figure 2: Structure of the Zn²⁺ -binding site at the C-terminus of the human S100B dimer. The subunits of the Zn²⁺-Ca²⁺-S100B are shown in blue and light-blue, Ca²⁺ ions are shown as yellow spheres, Zn²⁺ ions are shown as magenta spheres. The electron density (green) for coordinating residues and the Zn²⁺ ions are shown at level of 1.5σ. A) Zn²⁺ -binding site at pH 6.5. The Zn²⁺ is coordinated by His15, His25 from one subunit as well as by His85´

and Glu89´ from the second subunit. B) The Zn²⁺-binding site at pH 9.0. The Zn²⁺ ion is coordinated by four imidazole, His15, His25 from one subunit and from His85´, His90´ at the C-terminus of the second subunit. C) Zn²⁺ binding site of the pH 10.0 condition. The Zn²⁺

ion is coordinated by three histidine and one glutamate Glu89´, two His from one subunit His15, His25 and on from His85´the of second subunit. In all cases, the Zn²⁺ ion is coordinated in a tetrahedral manner, whereby His15, 25 and 85´ are always part of the Zn²⁺ coordination sphere. The fourth ligand at the very end of helixIV can be substituted either by His90´ or by Glu89´.

Figure 3: Structural alignment of the human Zn²⁺-Ca²⁺-S100B (light blue) with human Ca²⁺-S100B (red; PDB entry 2H61). Bound ions are shown as spheres, Zn²⁺ in light blue and Ca²⁺ in yellow. Human Zn²⁺-Ca²⁺-S100B (pH 10.0) exhibits an elongation in helix IV compared to the human Ca⁺²-S100B (red).

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Figure 4: Superposition of the S100B Zn²⁺-binding sites:

A-C: Superposition of the Zn²⁺-binding sites from S100A7 (light orange; PDB entry 2PSR) and the Cu²⁺-binding site of S100A12 (green; 1ODB) with the individual structures of this study, A: pH 6.5 yellow, B: pH 9.0 magenta and C: pH 10.0 light blue.

D: The positions of the coordinating ligands (H15, H25, H85´, and E90´) are similar within three obtained structures of this study, an exception is found for E90´ in the pH 9.0 Zn²⁺-Ca²⁺-S100B structure (Fig. B: magenta). The coordinating ligand H89´ from A7 and A12 (Fig. A-C) is substituted by the followed residue E90´. Ions are shown as colored spheres, according to their ligands.

Figure 5: Comparison of N-terminal S100-specific EF-hands of Zn²⁺-Ca²⁺-S100B at pH 10.0 (light blue) with the rat apo-S100B (yellow; PDB entry 1B4C), with human Ca²⁺ -S100B (red; PDB 2H61) and the human Cu²⁺-Ca²⁺-S100A12 (green; PDB entry 1ODB).

Ions are shown as spheres, Ca²⁺ as large spheres, Zn²⁺ and Cu²⁺ as smaller spheres. A:

Alignment of the apo-S100B (yellow) with the Zn²⁺-Ca²⁺-loaded S100B (light blue). B:

The alignment illustrates that there is almost no change in the S100-specific EF-hand, in the Zn²⁺-loaded (light-blue) compared to the Ca²⁺-loaded S100B (red), only His25 of the Zn²⁺-loaded structure (light-blue) is reoriented and points now towards the Zn²⁺ ion. C:

Alignment of the Zn²⁺-Ca²⁺-S100B (light-blue) with the Zn²⁺-Cu²⁺-S100A12 (green).

The residue Asp25 in the Cu²⁺-Ca²⁺-S100A12 (green) at the same position like the His25 in Zn²⁺-Ca²⁺-S100B (light-blue), are in an orientation enabling Zn²⁺ coordination.

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Figure 6: Increased α-helical content in Zn²⁺-Ca²⁺-loaded S100B. The secondary structure was analyzed by CD-spectroscopy, showing correctly folded S100B homodimers: solid line, Zn²⁺-Ca²⁺-S100B; dotted line, Ca²⁺-S100B.

Protein Technique Metal Ligands Geometry Ref.

Spectro-photometry Zn²⁺ Probably His &

Cys n.s. c

Table 1: List of known Zn²⁺ binding S100 proteins. n.s., not specified; h, human.

References: a, Randazzo et al., 2001; b, Koch et al., 2006 (submitted) ; c, Föhr et al., 1995, Fritz et al., 1998; d, Petrocchi et al., 1994; e, Schäfer et al., 2000; f, Kordowska et al., 1998; g, Brodersen et al., 1999; h, Kerkhoff et al., 1999b; i, Moroz et al., 2003; j, Dell´Angelica et al., 1995; k, Wilder et al.,2003; l, This work.

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aRfree was calculated against 5% of the total reflections omitted from the refinement.

Table 2: Data and refinement statistics.

Crystallization conditions pH 6.5 pH 9 pH 10

Space group P21 C2221 P21

No. of unique reflections 23032 15236 20781

Mean redundancy 4.0 6.5 3.5

interhelical angle (deg) interhelical distances (Å)

I-II II-III III-IV I-II II-III III-IV

2Human Zn²⁺-Ca²⁺-S100B, pH 6.5 137.4 99.6 105.5 15.5 11.6 15.7

2Human Zn²⁺-Ca²⁺-S100B, pH 9 137.7 99.9 107.0 15.4 11.8 15.0

2Human Zn²⁺-Ca²⁺-S100B, pH 10 138.0 100.5 106.0 15.4 11.7 15.0

3Human Ca²⁺-S100B, (pH 7.5) 136.9 98.9 103.8 15.5 11.6 15.7

4Rat Zn²⁺-Ca²⁺-S100B, (NMR pH 7.2) 130.2 128.5 139.6 14.6 11.0 8.5

4Rat apo-S100B, (NMR) 133.5 -142.1 -144.8 15.4 12.9 10.1

5Human Cu²⁺-Ca²⁺-S100A12 141.5 106.5 119.7 14.2 11.3 15.7

Table 3: Interhelical angles and distances were calculated using Interhlx (Yap, 1998).

Helices for Zn²⁺-Ca²⁺-S100B² (pH 6.5; 9.0; 10.0) and Ca²⁺-S100B³ structures were defined as helix I = 2-19, II =30-40, III = 51-61, and IV = 71-86. ⁴Calculated S100B NMR structure 1XYD (Wilder et al., 2003), and helices were defined helix I = 1-18, II

=29-39, III = 51-61, and IV = 70-84. ⁵Calculated S100A12 X-ray structure using PDB entry 1ODB (Moroz et al., 2003), and helices were defined helix I = 2-19, II =29-40, III

= 50-60, and IV = 70-89.

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Zn to ligand pH 6.5 pH 9 pH 10

His 15 Nε2 (Å) 2.04 2.03 2.01

His 25 Nε2 (Å) 2.05 1.88 1.94

His 85 Nε2 (Å) 2.26 2.10 2.0

His 90 Nε2 (Å) ^___ 1.91 ^___

Glu 89 O ε1 (Å) 2.06 ^___ 2.08

His 15 - His 25 (deg) 130.56 117.67° 115.38

His 15 - His 85 (deg) 106.55 106.26° 102.10

His 25 - His 85 (deg) 95.60 115.13° 99.79

His 90 - His 25 (deg) ^___ 107.55 ^___

Glu 89 - His 25 (deg) 122.64 ^___ 136.17

Table 4: Zn²⁺ distances and bound angles.

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4 C ONCLUSIONS

The focus of this study was to investigate the structure of human S100B in different metal-loaded states and its interaction with the surface membrane receptor RAGE (receptor for advanced glycation endproducts). Recombinant human S100B (hS100B) was expressed and purified to homogeneity in large quantities which allowed its following crystallization. For the first time, non-covalent multimeric species of hS100B were prepared from E. coli. Moreover, in independent experiments, the existence of these multimeric species could be demonstrated in human brain extracts. The crystallization of hS100B in the presence of calcium resulted in an octameric form of Ca²⁺-hS100B, further crystallization in presence of calcium and zinc at pH 6.5, 9.0 and 10.0 resultsed in comaparable structures of dimeric Zn²⁺-Ca²⁺-hS100B. Besides this, binding studies of human S100B and its discovered oligomeric species with the human RAGE receptor were carried out. This required the expression of the soluble domain of the RAGE (sRAGE). The receptor was expressed, purified and characterized, concomitant specific anti bodies against all the three domains of sRAGE (V-C1-C2) were constructed, to monitor the purification procedure as well as the results of the experiments.

4.1. Structure of the octameric S100B and the RAGE interaction with multimeric

Im Dokument Structure and function of the metal-binding protein S100B and its interaction with the receptor for advanced glycation end products (Seite 114-130)