Structure of the octameric S100B and the RAGE interaction with multimeric S100B species

In this study it has been shown that besides dimeric S100B also a major portion of multimeric forms of S100B are occurring in human brain, which was demonstrated by preparation of human brain samples. Notably the most prominent forms of the S100B multimers were the tetrameric and octameric species. Such multimeric species from human brain are here described for the first time (3.3.6. Fig.1). Suchlike non-covalent multimeric species were also detected upon high yield expression of human S100B in E.

coli (3.1.5. Fig.1). Upon Ca²⁺ binding the structure of S100B opens and exposes a protein-protein interaction site. The interaction site of S100B provides hydrophobic and polar residues which are required for high affinity binding of target proteins like p53 (Rustandi et al., 2000b), CapZ (Inman et al., 2002b; McClintock and Shaw, 2003),

NDR-kinase (Bhattacharya et al., 2003) or RAGE (Donato, 2003). Far UV-CD spectroscopic analysis of these multimeric species confirmed that tetrameric, hexameric and octameric human Ca²⁺-S100B possess the same secondary structure (3.3.6. Fig.2 B). The content of helix was 64% and fits very well to the octameric X-ray structural data with 63% α-helix, as determined with DSSP (Kabsch and Sander, 1983). These results demonstrate that the human Ca²⁺-S100B multimers are fully folded and functional. Functionality of the Ca²⁺-dependent conformational change in the different S100B species was verified by binding to the hydrophobic phenyl-Sepharose matrix in the presence of Ca²⁺ and elution from the matrix with EDTA. Thus, the different multimeric hS100B species are fully functional and may serve as Ca²⁺ sensor proteins like the human S100B dimer.

Moreover, we were able to show that once formed multimeric S100B species are stable in solution and does not show any kind of interconversion (3.3.6. Fig.2 A). The formation of multimers and their stability is obtained independently from disulfide bridge formation. This was demonstrated by structural analysis of the octameric human Ca²⁺-S100B (3.3.6. Fig.3 A), as well as by native SDS-Page analysis. (3.3.6. Fig.2 C).

The solute multimeric species tetra-, hexa- or octameric are also stable even in presence of dioxygen. This property is crucial for proper folding and multimer formation in the oxidizing, extracellular space, because a disulfide formation will be associated with dramatic changes in the structural arrangement of the several subunits. For such multimeric species of S100-proteins it has been proposed that multimerization may play a major role in extracellular and intracellular signaling. Very recently it has been shown for S100A4 that only the multimeric forms exhibit neurite sprouting activity (Kiryushko et al., 2006). In the case for Ca²⁺-S100A8/A9 it was shown that a Ca²⁺ induced tetramer formation is required for its activity, which promote the formation of microtubules (1XK4, http://www.rcsb.org, (Leukert et al., 2006). For Ca²⁺-S100A12 it was shown that additional Ca²⁺ ions, besides the EF-hand Ca²⁺ ions, triggers the formation of a hexameric species which was proposed to be one active species in RAGE receptor signaling (Moroz et al., 2002). The crystal structure of human Ca²⁺-S100B obtained in this study revealed an octameric organization whereby four dimers form a tight assembly. There is no similarity in the assembly with the S100A8/A9 (1XK4) tetramer or the S100A12 hexamer (Moroz et al., 2002). Nevertheless, one common feature in the all three multimeric structures of S100 proteins is the occurrence of additional Ca²⁺ ions, distinct of those bound in the EF-hands. These additional Ca²⁺ ions are bound at the interface of the subunits most likely stabilizing the larger octameric structures. This

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finding implies that high Ca²⁺ concentrations might be required for the formation of the S100 protein multimers. In particular high Ca²⁺ concentrations are present in the extracellular space where S100A8/A9, S100A12, S100B act as pro-inflammatory signaling molecules via the receptor RAGE (Donato, 2003).

Efforts to crystallize a multimeric species of human S100B to elucidate multimer organization results in the structure of an octameric human S100B that is shown for the first time in this work. The approximate size of 2050 Ų of contact areas, built by hydrophilic and hydrophobic interactions, between eight subunits indicating that this formation of four dimers to one octamer is not a crystallographic artifact. This has been also proven by calculation of the self-rotation function, indicating the presence of several two-fold non-crystallographic axes (3.1.5. Fig.4). The N-terminal helices (HI) of the 8 subunits outline the central cavity of the octameric structure (3.3.6 Fig.3 A/B), whereas helices HII reside at the top and the bottom of the octameric molecule. The C-terminal EF-hands of all subunits encompassing helices HIII and HIV are exposed to the solvent at the side of the octameric structure. This arrangement leads to an accumulation of hydrophobic surface areas, which combine to a large putative target-binding site (3.3.6.

Fig.3 C). Remarkably, in a tetrameric or octameric human S100B two target protein interaction sites get in close proximity on one face of the molecule. Binding of tetrameric or octameric human S100B will possibly approximate target proteins like RAGE in such a way that intermolecular interactions between the target molecules become more likely.

It has been shown that S100B can be secreted from astrocytes (Pinto et al., 2000) and gliablastomacells to the extracellular matrix by an increase of calcium and a decrease of zinc (Davey et al., 2001). In the extracellular space, the calcium concentration is on average 10.000 fold higher then intracellularly, so one can assume that S100B appear only in a calcium-loaded state instantly it reaches the extracellular space. It was found that S100B acts extracellularly as a proinflammatory cytokine in vascular smooth muscle cells or as a trophic factor on glia cells via the receptor RAGE (Huttunen et al., 2000;

Reddy et al., 2006). Immunoassay analysis with the different purified S100B species showed specific and strong binding to sRAGE even in presence of competitors like BSA or milk powder. In addition to this, ELISA studies with the same types of multimeric S100B showed comparable results as it was shown in the immunoassays. All of the multimeric S100B species show a higher binding affinity to sRAGE than dimeric S100B (3.3.6. Fig. 6 A/B). To investigate this, we analyzed the binding of dimeric and tetrameric S100B to RAGE, in real time by surface plasmon resonance. The results

showed for the first time that the interaction of S100B with RAGE is strictly Ca²⁺ -dependent and that dimeric S100B recognizes sRAGE basically only as one species on the sensor chip (3.3.6. Fig.7 C). Tetrameric S100B binds to GST-sRAGE with higher affinity (3.3.6. Fig.7 B) than dimeric S100B. The dissociation constants of Kd 1 = 2.3 μM and Kd 2 = 0.4 μM show that S100B tetramer bound to sRAGE with 10 to 50-fold higher affinity than S100B dimer. The multiple binding sites of tetrameric S100B for RAGE possibly will trigger receptor multimerization, which has been established as a general mechanism for the initiation of signal transduction. Many cell-surface receptors are activated by such a ligand induced multimerization process such as toll like receptors (de Bouteiller et al., 2005; Hu et al., 2004) or tumor necrosis factor α receptor (Bazzoni et al., 1995). Additional docking studies using the V-domain model for RAGE and the structure of human S100B tetramer (chains A-D) revealed a strong clustering of possible docking sites at the top of the V-domain dimer model where the long variable loops of the Ig domain are located (3.3.7. Fig8). Some high scoring docking results showed that only one S100B dimer interacts mainly with one V-domain molecule. These results are in agreement with the observation that S100B dimer binds to sRAGE in surface plasmon resonance studies. RAGE activation by S100B represents the major pro-inflammatory pathway in neurodegenerative or auto-immune diseases (Arancio et al., 2004; Yan et al., 1996; Yan et al., 2003). The structure and binding data of this study provide insights into the mechanisms of S100B induced RAGE multimerization. The proposed binding model originating from molecular modeling and docking calculations revealed highly complementary surfaces for tetrameric S100B and a RAGE V-domain dimer.

We propose that tetrameric and octameric species of S100B can provoke an accumulation of RAGE receptors. Formation of multimeric S100B may occur during secretion in the vesicles. After secretion to the extracellular space, the multimeric S100B species binds to RAGE. Interestingly, the isoform DN-RAGE (dominant negative RAGE) which is devoid of the intracellular domain prevents activation of wild-type RAGE by its ligands and thereby leads to the blockade of the signal transduction into the cytoplasm. (Schmidt and Stern, 2001). These findings indicate that RAGE receptor multimerization is a crucial step in signal transduction, and this multimerization can be assisted by the multimeric species of S100B that was discovered in this study.

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