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• W2003739799 abstract "Article26 July 2007free access Structural and functional insights into RAGE activation by multimeric S100B Thorsten Ostendorp Thorsten Ostendorp Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Estelle Leclerc Estelle Leclerc Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zürich, Switzerland Search for more papers by this author Arnaud Galichet Arnaud Galichet Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zürich, Switzerland Search for more papers by this author Michael Koch Michael Koch Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Nina Demling Nina Demling Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany Search for more papers by this author Bernd Weigle Bernd Weigle Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany Search for more papers by this author Claus W Heizmann Claus W Heizmann Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zürich, Switzerland Search for more papers by this author Peter MH Kroneck Peter MH Kroneck Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Günter Fritz Corresponding Author Günter Fritz Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Thorsten Ostendorp Thorsten Ostendorp Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Estelle Leclerc Estelle Leclerc Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zürich, Switzerland Search for more papers by this author Arnaud Galichet Arnaud Galichet Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zürich, Switzerland Search for more papers by this author Michael Koch Michael Koch Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Nina Demling Nina Demling Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany Search for more papers by this author Bernd Weigle Bernd Weigle Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany Search for more papers by this author Claus W Heizmann Claus W Heizmann Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zürich, Switzerland Search for more papers by this author Peter MH Kroneck Peter MH Kroneck Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Günter Fritz Corresponding Author Günter Fritz Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany Search for more papers by this author Author Information Thorsten Ostendorp1, Estelle Leclerc2, Arnaud Galichet2, Michael Koch1, Nina Demling3, Bernd Weigle3, Claus W Heizmann2, Peter MH Kroneck1 and Günter Fritz 1 1Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, Germany 2Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zürich, Switzerland 3Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany *Corresponding author. Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Universitätsstrasse 10, Konstanz 78457, Germany. Tel.: +49 7531 88 3205; Fax: +49 7531 88 2966; E-mail: [email protected] The EMBO Journal (2007)26:3868-3878https://doi.org/10.1038/sj.emboj.7601805 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF-hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X-ray structure of human Ca2+-loaded S100B was determined at 1.9 Å resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size-exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V-domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation. Introduction S100B is a member of the S100 protein family which represents the largest subgroup of EF-hand Ca2+-binding proteins (Heizmann et al, 2002; Donato, 2003; Marenholz et al, 2004). The individual members of the S100 family show tissue- and cell type-specific expression patterns and regulate processes such as cell growth and motility, transcription and differentiation. S100 proteins except S100G (calbindin D9K) mostly form homo- and heterodimers. Each subunit consists of two helix-loop-helix (EF-hand) motifs connected by a central linker or so-called hinge region. The C-terminal canonical EF-hand motif is composed of 12 amino acids, whereas the N-terminal S100-specific EF-hand comprises 14 residues. The S100 proteins undergo a conformational change upon Ca2+ binding, which leads to the exposure of a protein–protein interaction site (Ikura and Ames, 2006). S100B is mainly synthesised by astrocytes in human brain and modulates long-term synaptic plasticity (Nishiyama et al, 2002). Intracellularly, S100B modulates microtubule assembly and regulates the cell cycle by interacting with transcription factors such as p53. Upon changes in intracellular Zn2+ and Ca2+ concentrations, S100B is secreted from glia cells to the extracellular space, where it exhibits cytokine-like functions (Davey et al, 2001). The action of S100B is strongly dependent on its extracellular concentration. At nanomolar level, S100B is neuroprotective, whereas it promotes apoptosis at micromolar concentration (Huttunen et al, 2000; Businaro et al, 2006). Such high extracellular levels are detected after traumatic brain injury or in neurodegenerative disorders like Down's Syndrome, Alzheimer disease or encephalomyelitis (Griffin et al, 1998; Mrak and Griffin, 2001). Both trophic and toxic effects of extracellular S100B are mediated in the brain by the receptor RAGE (receptor for advanced glycation end products) (Hofmann et al, 1999), which was initially described as a receptor for glucose-modified proteins, involved in the development of chronic inflammations in diabetes patients (Neeper et al, 1992). RAGE is a member of the immunoglobulin (Ig) superfamily of cell-surface receptors and shares homology with neuronal cell adhesion molecules like NCAM or axonin (Freigang et al, 2000; Soroka et al, 2003). RAGE consists of an extracellular moiety, a single transmembrane spanning helix and a short cytosolic domain which is required for signal transduction. The extracellular moiety includes one N-terminal V-type and two C-type Ig domains. Increased RAGE expression is observed in human diseases including diabetes, Alzheimer disease and inflammations (Ramasamy et al, 2005). The levels of RAGE rise upon stimulation through extracellular ligands, resulting in an increasing cellular response and establishment of a chronic inflammation. To disrupt this cycle, detailed knowledge of the nature of the ligand–receptor interaction is required. Although comprehensive structural information on S100B is available, the known structures reflect only the intracellular state of S100B. Extracellularly, S100B can form disulphide bonds required for its neurite extension activity (Selinfreund et al, 1991; Kiryushko et al, 2006), but not for the pro-inflammatory activity of S100B (Koppal et al, 2001). The presence of disulphide bonds suggests the formation of covalently linked multimers, but leaves the state of pro-inflammatory acting S100B questionable. Here, we report the detection of noncovalent S100B multimers in human brain. Stable S100B multimers were purified from Escherichia coli and bound with high affinity to the extracellular domain of RAGE (sRAGE). The crystal structure of human S100B revealed a noncovalent octameric assembly consisting of two tetramers. On the basis of these structural data, binding studies and in silico docking calculations, we propose that tetrameric S100B activates RAGE through receptor dimerisation. Results S100B multimers in human brain The presence of multimeric S100B in human brain extract was demonstrated by size-exclusion chromatography (SEC). The elution profile of S100B from SEC was monitored by an ELISA test specific for S100B. In the presence of Ca2+, S100B elutes in three superimposing peaks corresponding to tetramer, hexamer and octamer, whereas in the presence of EDTA, S100B elutes in two major peaks corresponding to tetramer and dimer (Figure 1B). The high molecular mass peaks of S100B, in the presence of Ca2+, might represent higher multimers such as hexamer and octamer, but could also represent complexes of Ca2+-loaded S100B dimer or S100B tetramer with target proteins. However, calculating the molecular masses of complexes of known target proteins with S100B dimer reveals that most complexes are much larger than the observed peaks (Supplementary Figure I). We therefore conclude that the observed peaks mainly represent the larger multimers such as hexamer and octamer of S100B. Figure 1.Detection and molecular mass analysis of S100B multimeric species. Human brain extracts were analysed by SEC. (A) Elution profile of S100B from human brain extract in the presence of EDTA. (B) Elution profile of S100B in human brain extracts in the presence of Ca2+. The elution volumes of standards (cytochrome c 12.4 kDa, carboanhydrase 29 kDa, conalbumin 76 kDa) and the elution volumes of isolated recombinant S100B species from the same column are indicated. (C) Elution profiles of isolated recombinant S100B species (dimer ▵; tetramer •, hexamer □, octamer ▾) from analytical SEC. The elution volumes of standards are indicated. (D) Analysis of the isolated S100B multimers by analytical ultracentrifugation. The fits for molecular mass distribution for S100B dimer (−▵−), tetramer (–•–), hexamer (–□–) and octamer (–▾–) samples are shown. Download figure Download PowerPoint In the presence of EDTA, which abolishes S100B–target protein interactions, about 50% of the protein elutes as tetramer and 50% as dimer (Figure 1A). The elution profiles document the presence of oligomers of S100B in the brain and reveal that multimers represent a large portion of S100B in human brain. Biochemical characterisation of human recombinant multimeric S100B Purification of recombinant human S100B from E. coli yields dimeric, tetrameric, hexameric and octameric S100B (Ostendorp et al, 2005). These multimers were stable and isolated multimers eluted as one major peak from SEC (Figure 1C). No equilibrium between the different S100B species was observed. Upon freezing and thawing, octameric and hexameric S100B partially dissociated into tetramers and dimers. In contrast, tetrameric S100B was stable and did not dissociate further into dimers. The molecular masses of the different multimeric species were judged from a calibration of the column with proteins of known size. The mass of the isolated S100B species was further analysed by analytical ultracentrifugation (Figure 1D). Analysis of the sedimentation behaviour revealed masses of 21 kDa for dimeric, 41 kDa for tetrameric, 61 kDa for hexameric and 82 kDa for octameric S100B, which are in good agreement with the calculated masses (dimer 21.4 kDa, tetramer 42.8 kDa, hexamer 63.2 kDa and octamer 85.6 kDa). The Ca2+-induced conformational change of dimeric S100B has been well characterised by NMR and X-ray crystallographic studies (Kilby et al, 1996; Matsumura et al, 1998; Drohat et al, 1998a, 1999). These studies showed that dimeric S100B undergoes a conformational change upon Ca2+ binding, leading to the exposure of a hydrophobic patch at the surface of the molecule. To test whether the isolated multimers undergo, like dimeric S100B, a Ca2+-dependent conformational change, the different species were bound in the presence of Ca2+ to hydrophobic phenyl–Sepharose matrix and eluted from the matrix with EDTA. Subsequent analysis of the eluting protein by analytical SEC showed that the multimers did not dissociate on the phenyl–Sepharose column. Thus, the different multimeric S100B species are fully functional as Ca2+-sensor proteins like the S100B dimer. The secondary structures of the dimeric, tetrameric and octameric S100B were analysed by far UV-CD spectroscopy. The CD spectra of the different species were virtually identical (Supplementary Figure IVA) and showed that all S100B species were correctly and presumably identically folded. The content of α-helix as determined by CD spectroscopy was 64% and fits very well the X-ray structural data with 63% α-helix as determined by DSSP (Kabsch and Sander, 1983). These results demonstrate that the S100B multimers are fully folded and functional. Previous studies revealed that a disulphide linked form of S100B is required for its neurotrophic activity (Selinfreund et al, 1991), but not for the induction of an inflammatory response in glia cells (Koppal et al, 2001). The S100B multimers studied here were not bridged by intermolecular disulphide bonds as shown by SDS–PAGE under nonreducing conditions (Supplementary Figure IVB). Furthermore, we expressed and purified a Cys → Ser mutant of S100B, which gave the same distribution of dimeric, tetrameric, hexameric and octameric S100B in SEC experiments. This demonstrates that neither the formation of the multimers nor their stability is dependent on disulphide bridges. The X-ray structure reported here confirms our data from biochemical studies. Tetrameric S100B promotes stronger cellular growth and survival of HeLa cells S100B is released by cells into the intracellular space and can directly affect cells (Davey et al, 2001). To examine the role of S100B multimerisation in cellular functions, we treated HeLa cells with both S100B dimers and tetramers. We had observed previously that hexameric and octameric S100B partially dissociated, respectively, into tetramer and dimer upon freezing and thawing. Therefore, we tested the stability of the different S100B species in cell culture media at 37°C before the cell activation assays. The different species of S100B were analysed by SEC on a Superdex 75 column. After 24 h, octameric and hexameric S100B were dissociated into tetramers and dimers and only ca. 20% of the original amount of the largest species was still present. In contrast, tetrameric S100B remained intact up to 12 h and after 24 h 80% of the original tetramer was still observed under the same conditions. S100B dimer did not form any larger oligomers even after 48 h at 37°C in cell culture medium. Therefore, we used only dimeric and tetrameric S100B for cell activation assays. To clearly resolve the effect of dimeric and tetrameric S100B, we used the same mass concentration of protein (100 μg/ml), that is, the molar concentration of S100 tetramer (2.35 μM) was just half of the concentration of S100B dimer (4.7 μM) in the assays. Nevertheless, exogenous tetrameric S100B increased cell proliferation already after 8 h, whereas cells treated with the dimeric form of the protein only showed increased proliferation after 24 h (Figure 2A). Similarly, S100B tetramers-treated cells had decreased caspase 3/7 activities compared with control or S100B dimers-treated cells (Figure 2B). These results corroborate that the multimerisation state of S100B strongly affects its function. The stronger activation of cell proliferation and cell survival by S100B tetramer, although the molar concentration was only half of that of the S100B dimer, can be readily explained by stronger binding of S100B tetramer to RAGE as observed in surface plasmon resonance (SPR) experiments (see below). Figure 2.S100B tetramers activate HeLa cells differently than S100B dimers. (A) Effect of S100B dimers (100 μg/ml, 4.7 μM) and tetramers (100 μg/ml, 2.35 μM) on cell proliferation was estimated after 8 and 24 h treatment by MTT assay (n=8). (B) Effector caspase 3 and 7 activities were similarly monitored after 8-h treatment with PBS alone or S100B species (n=4). All data are expressed as means±s.d. Statistical analysis was performed using one-way analysis of variance, followed by Student's t-test. Significance was considered as P⩽0.0001 (**) or P⩽0.001 (*) versus the control group. Download figure Download PowerPoint Crystal structure of S100B Overall structure. Human recombinant S100B in the Ca2+-loaded state was successfully crystallised (Ostendorp et al, 2005) and the X-ray structure was refined to 1.9 Å resolution. The crystals grew from protein precipitates to a final size of 0.2 × 0.4 × 0.4 mm. The space group is P21 with unit cell parameters, a=63.4 Å, b=81.6 Å, c=71.5 Å, α=γ=90°, β=107°. The structure is well defined by the electron density except four side chains located at the protein surface and two residues in the C-terminal regions of the S100B subunits could not be resolved. The final model contains 728 amino-acid residues, 18 Ca2+ ions, one PEG400 and 713 water molecules, and converged to a Rcryst of 17.4% and Rfree of 24.0% using Refmac (Murshudov et al, 1997) for final refinement (Table I). The asymmetric unit contains four S100B dimers, which are related by an approximate noncrystallographic 222 symmetry (Figure 3A). This approximate 222 symmetry is very close to 180° for the pair of dimers AB–CD (179°) and EF–GH (178°), but exhibits lower symmetry for the AB–GH (174°) and CD–EF (172°) pairs, respectively (Supplementary Figure II). This difference in the pairings is also observed in the r.m.s.d.s, which are 0.91 and 0.59 Å for the AB–CD and EF–GH pairs, respectively, whereas the AB–GH and CD–EF pairs exhibit in each case higher r.m.s.d.s of 1.01 and 0.86 Å. These differences suggest that the dimers AB and CD form one tetramer, whereas the other tetramer is formed by the dimers of chains EF and GH. This assignment of two dimers to a tetramer is corroborated by the analysis of the interdimer interfaces, which show that for example, the dimers AB and CD cover a larger surface (1540 Å2) than the dimers AB and GH (1230 Å2). Figure 3.Overall structure of the S100B octamer. (A) Arrangement of eight S100B subunits (A–H) in the octamer. The view is down one noncrystallographic two-fold axis. The second noncrystallographic two-fold axis is vertical, and the second noncrystallographic two-fold axis is horizontal. Subunits AB are shown in green, CD in cyan, EF in magenta and GH in yellow. The Ca2+ ions are shown as spheres. Sixteen Ca2+ ions located in the EF-hand are shown in red and two Ca2+ ions bound at the subunit interface are shown in orange. (B) Surface representation of the octamer showing the tight assembly of the subunits. (C) Hydrophobic surface of S100B octamer. The side view of S100B shows the large hydrophobic surface (orange) formed by two adjacent S100B dimeric units (AB and CD) of the octamer. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Wavelength (Å) 1.54 Resolution limit (Å) 30–1.90 (2.0–1.9)a I/σ 15.9 (4.0)a Space group, Unit cell (Å) P21; a=63.4, b=81.6, c=71.5, α=γ=90°, β=107° Completeness (%) 98.9 (94.1) Multiplicity 3.6 Rmrged-F (%)b 7.8 (31.5)a Resolution range 30–1.9 Rcryst (%)c 17.4 (19.9)a Rfree (%)d 24.0 (25.5)a RMS bond length (Å) 0.017 RMS bond angles (deg) 1.6 a The numbers in parentheses are the statistics for the highest resolution shell. b Rmrgd-F (%)=Σ (∣AIh,P−AIh,Q∣)/(0.5*ΣAIh,P+AIh,Q). c Rcryst=Σhkl(∣F(obs)∣−∣F(calc)∣)/Σhkl∣F(obs)∣. d Rfree=Σhkl(∣F(obs)∣−∣F(calc)∣)/Σhkl∣F(obs)∣, where 5% of the observed reflections are not used for refinement. The S100B octamer has a donut-like shape, with a diameter of 82 Å, containing a central cavity with an approximate diameter of 10 Å (Figure 3B). The four dimers in the assembly are very similar to the so far determined 3D structures of S100B, like for example, the crystal structure of bovine S100B (pdb code 1MHO) (Matsumura et al, 1998), as illustrated by an r.m.s.d. of 1.0 Å for 174 Cα positions. Each subunit in the octamer consists of two pairs of EF-hands connected by a flexible linker region. The Ca2+ ions in the EF-hands of the subunits are well defined by the electron density and are coordinated by the sidechains of Asp and Glu residues and by backbone carbonyl oxygen atoms. The X-ray structure of human S100B revealed structural changes induced by Ca2+ binding similar to the structures of Ca2+-loaded rat and bovine S100B (Matsumura et al, 1998; Drohat et al, 1998a). Comparison with structures of Ca2+-free S100B from rat (Drohat et al, 1999) reveals that Ca2+ binding induces a rather small conformational change in the N-terminal S100-specific EF-hand, but a large change in the canonical C-terminal EF-hand. Helix HIII moves by about 90°, changing the orientation of helix HIII towards helix HIV from an antiparallel to a perpendicular orientation. The N-terminal helices (HI) of the eight subunits outline the central cavity of the octameric structure (Figure 3A and 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 (Figure 3C). To get an idea of the structural changes in the tetramer induced by Ca2+ binding, we built a model of Ca2+-free S100B tetramer using the structure of rat S100B in the Ca2+-free form (Drohat et al, 1999). In the model, the conformation of helix HIV had to be slightly adjusted to form the interdimer contact of the tetramer. Surface analysis of the Ca2+-free tetramer model showed that the large hydrophobic area observed in the structure of Ca2+-loaded S100B tetramer (Figure 3C) was absent (data not shown). As expected, in the model of Ca2+-free tetramer, a part of the hydrophobic area was covered by helices HIII of the C-terminal EF-hands. The hydrophobic patch observed in the centre between the two dimers was masked in the Ca2+-free form by residues of the linker regions. Thus, conformational changes of helices HIII and of the linker regions lead to the exposure of hydrophobic patches, which combine to the large area observed in the structure. Remarkably, in a tetrameric or octameric S100B, two target protein interaction sites get in close proximity on one face of the molecule. Binding of tetrameric or octameric S100B will possibly approach target proteins in such a way that intermolecular interactions between the target molecules become likely. In contrast, the two target interaction sites in dimeric S100B reside on opposite sides of the molecule (Rustandi et al, 2000; Inman et al, 2002; Bhattacharya et al, 2003; McClintock and Shaw, 2003), making intermolecular interactions between the bound target molecules unlikely. Intermolecular interactions of the S100B subunits in the octamer The size of the buried area at the interface between two dimers (subunits A–B, C–D) forming a tetramer (A–B–C–D) is 1540 Å2 which is in the typical range (1600±400 Å2) for protein–protein complexes (Lo Conte et al, 1999). The residues at the interface between dimers AB and CD, as well as between dimers EF and GH provide a number of hydrophobic contacts as well as five hydrogen bonds (Figure 4A–C). Assembly of the two tetramers ABCD and EFGH to the octamer buries a total surface area of ca. 2500 Å2. The interface is formed by a number of hydrophobic and polar interactions: for example, the hydrophobic residues Ile11 of subunit D and Cys84, Phe87 and Phe88 of subunit C shape a pocket which embeds the residues Phe87 and Phe88 of subunit E. At the interface between the two tetramers, two additional binding sites for Ca2+ were identified in the electron density difference maps. The geometry and bond distances as analysed by the program WASP (Nayal and Di Cera, 1996) and the electron density are in agreement with Ca2+ bound to the sites. Each Ca2+ ion is coordinated by residues from two different subunits and water molecules. Ser41 and Leu44 from one subunit (B or E) provide backbone carbonyl oxygens, whereas Asp12 from subunit H or D provides the sidechain for coordination of the Ca2+. The coordinated water molecules are stabilised by a hydrogen bond network around the binding site (Figure 5). Such additional Ca2+ ions at the interface of subunits were also observed in the hexameric structure of S100A12 (Moroz et al, 2002). The contact sites between the dimeric units in the S100B octamer structure show a rather high geometric surface complementarity, which most likely contribute to the stability of the octameric complex. Shape complementarity can be quantitated by defining a shape correlation statistic (Sc) that measures the degree of geometric match between two juxta-posed surfaces (Lawrence and Colman, 1993). Interfaces with perfect fits have Sc=1, whereas interfaces that are topographically uncorrelated have Sc values close to zero. The high shape correlation statistics (Sc values) of 0.68 for the interface formed by two dimers (AB+CD) are in the same range like those for S100B target peptide complexes (Rustandi et al, 2000; Inman et al, 2002; Bhattacharya et al, 2003; McClintock and Shaw, 2003), which gave an average value of 0.62±0.09. The Sc value of 0.76 for the interface between two S100B tetramers (ABCD+EFGH) is even higher than in antibody/antigen complexes which exhibit typical values of 0.64–0.68 (Lawrence and Colman, 1993). Figure 4.Interactions of subunits in S100B octamer. (A) Subunit contact formed by two antiparallel oriented helices of subunits A, B, C and D. Hydrogen bounds and salt bridges are formed between Asp12B and Lys5C, Asp12C and Lys5B, His 15B and His42D, and between His42A and His15C. Additional hydrophobic contacts are formed by Val8 of subunits B and C. (B) Interactions between subunits C, D, E and F. There are hydrophobic contacts between Val8D and Phe43E, as well as ionic interactions between by His42C and Asp12F. (C) Hydrophobic interactions formed by C-terminal residues. The subunits AB–GH are linked by a hydrophobic knot formed by residues Phe87B and Phe88B and Ile11G, Cys84H, Phe87H and Phe88H. Download figure Download PowerPoint Figure 5.Intersubunit Ca2+-binding site. View of the CD–EF intersubunit Ca2+-binding site. Ca2+ coordination and hydrogen bonds are indicated by dashed lines. Ca2+ to oxygen distances are in the range of 2.2–2.6 Å. The Ca2+ (orange sphere) is coordinated in a distorted octahedral manner by the side chain of Asp 12D, backbone carbonyls from Ser41E and Leu44E and the water molecules W30, W31 and W32 (red spheres). A 2fo−fc electron density (blue) is shown countered at 1.5 σ and a fo-fc map, where the Ca2+ was omitted is countered at 7.0 σ (red). Download figure Download PowerPoint To validate the interdimer and intertetramer interfaces, we compared the sequences of S100B from nine species. The residues involved in the interdimer and intertetramer contacts are all strictly conserved in S100B. Remarkably, S100B exhibits the highest grade of conservation among all S100 proteins with 96% identity over 91 residues (Supplementary Figure IIIA). Furthermore, we constructed two S100B variants where Asp12 or Phe87/Phe88 were exchanged to Ala. Both variants were isolated by the same procedure as S100B wild type. SEC showed that recombinant S100B-D12A and S100B-F87AF88A form mainly dimer (87%/81%) and much less tetramer (9%/10%) or hexamer (3%/9%) compared to recombinant S100B wild type (61±8 dimer, 25±7 tetramer, 11±3 hexamer, 3±1.4 octamer; n=4) (data are summarised in Supplementary Table I). Octameric S100B was not detected for both variants. These results strongly support that the crystallographically observed interfaces reflect the situation in solution and are not an artefact of crystal packing. S100B binding to sRAGE The binding modes of S100B to intracellular target proteins have been characterised in great detail. Upon Ca2+ binding, the structure opens and exposes a protein–protein interaction site. The interaction site of S100B provides hydrophobic" @default.
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• W2003739799 date "2007-07-26" @default.
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• W2003739799 title "Structural and functional insights into RAGE activation by multimeric S100B" @default.
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