The C-terminus and linker region of S100B exert dual control on protein-protein interactions with TRTK-12

S100B, an EF-hand calcium-binding protein composed of two S100beta monomers, undergoes a calcium-dependent conformational change that provides a surface for target interactions. In this study, the calcium-sensitive S100B-binding epitope TRTK-12 has been used to probe the contributions of the linker and C-terminal regions of S100B to protein-protein interactions. These contributions were quantified using C-terminal mutant S100B proteins lacking the C-terminal seven (S100B85stop) or nine (S100B83stop) residues or containing alanine substitutions at Phe87 (F87A), Phe88 (F88A), or both (F8788A). Both F8788A and F88A bound TRTK-12 less tightly (K(d) = 1.85 +/- 0.02 and 0.97 +/- 0.08 microM, respectively) than the wild-type protein (K(d) = 0.27 +/- 0.03 microM, DeltaG = -37.2 kJ/mol), indicating these residues are important for TRTK-12 interaction. The truncated S100B proteins bound TRTK-12 much more weakly (K(d) = 659.7 +/- 119.3 microM, DeltaG = -17.9 kJ/mol), indicating the linker region contributed about 50% to the binding of TRTK-12, while the C-terminus contributed the remaining 50% of the binding energy. Based on mutagenesis and NMR chemical shift studies, a comparison with known S100-target protein complexes showed the S100B-TRTK-12 complex has the strongest resemblance to the S100A10-annexin II interaction.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.     

Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.     

Demonstration of Heterodimer Formation between S100B and S100A6 in the Yeast Two-Hybrid System and Human Melanoma

S100B (S100β) and S100A6 (calcyclin) are two 10-kDa Ca²⁺- and Zn²⁺-binding proteins coexpressed in melanoma and cell-cycle regulated. These proteins are members of the S100 subfamily and are thought to exert their function through interaction with intracellular target proteins. In order to search for potential target proteins interacting with S100B, we used a yeast two-hybrid strategy with human S100B as bait to screen a human brain cDNA library. The fusion proteins interacting with the S100B bait were identified as S100B, S100A1, and S100A6. This indicates the potential of S100B to form homodimers and heterodimers with other members of the S100 subfamily. By Northern and Western blotting, S100B and S100A6 were shown to be expressed at high levels in a panel of human melanoma cell lines. S100B and S100A6 were coimmunoprecipitated from melanoma cell lysates in the presence of 100 μM Zn²⁺. Confocal microscopy demonstrated that both proteins were distributed throughout the cytoplasm and concentrated in the nucleus. The demonstration of an association and colocalization of S100B and S100A6 in melanoma supports the possibility that an S100B/S100A6 heterodimer plays a functional role in these cells.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

S100A1 and S100B interactions with annexins

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.     

Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100 target specificity and activation of the kinase

NDR, a nuclear serine/threonine kinase, belongs to the subfamily of Dbf2 kinases that is critical to the morphology and proliferation of cells. The activity of NDR kinase is modulated in a Ca(2+)/S100B-dependent manner by phosphorylation of Ser281 in the catalytic domain and Thr444 in the C-terminal regulatory domain. S100B, which is a member of the S100 subfamily of EF-hand proteins, binds to a basic/hydrophobic sequence at the junction of the N-terminal regulatory and catalytic domains (NDR(62-87)). Unlike calmodulin-dependent kinases, regulation of NDR by S100B is not associated with direct autoinhibition of the active site, but rather involves a conformational change in the catalytic domain triggered by Ca(2+)/S100B binding to the junction region. To gain further insight into the mechanism of activation of the kinase, studies have been carried out on Ca(2+)/S100B in complex with the intact N-terminal regulatory domain, NDR(1-87). Multidimensional heteronuclear NMR analysis showed that the binding mode and stoichiometry of a peptide fragment of NDR (NDR(62-87)) is the same as for the intact N-terminal regulatory domain. The solution structure of Ca(2+)/S100B and NDR(62-87) has been determined. One target molecule is found to associate with each subunit of the S100B dimer. The peptide adopts three turns of helix in the bound state, and the complex is stabilized by both hydrophobic and electrostatic interactions. These structural studies, in combination with available biochemical data, have been used to develop a model for calcium-induced activation of NDR kinase by S100B.     

Molecular interface of S100A8 with cytochrome b558 and NADPH oxidase activation

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b(558); and (iii) to determine the S100A8 consensus site involved in cytochrome b(558)/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91(phox) and p22(phox). Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b(558) suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic (87)HEES(90) amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b(558) and then in the phagocyte NADPH oxidase activation.     

Identification of calcium-independent and calcium-enhanced binding between S100B and the dopamine D2 receptor

S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and exposes a hydrophobic protein-binding surface. Recently S100B was identified as a binding partner of the dopamine D2 receptor in a bacterial two-hybrid screen involving the third intracellular loop (IC3). The low in vivo calcium concentration in bacteria (100-300 nM) suggests this interaction may occur in the absence of calcium. In this work the calcium-sensitive ability for S100B to recruit the IC3 of the dopamine D2 receptor was examined, and regions in both proteins required for complex formation were identified. Peptide array experiments identified the C-terminal 58 residues of the IC3 (IC3-C58) as the major interacting site for S100B. These experiments along with pull-down assays showed the IC3 interacts with S100B in the absence and presence of calcium. (1)H-(15)N HSQC experiments were used to identify residues, primarily in helices III and IV, utilized in the IC3-C58 interaction. NMR titration data indicated that although an interaction between apo-S100B and IC3-C58 occurs without calcium, the binding was enhanced more than 100-fold upon calcium binding. Further, it was established that shorter regions within IC3-C58 comprising its N- and C-terminal halves had diminished binding to Ca(2+)-S100B and did not display any observable affinity in the absence of calcium. This indicates that residue or structural components within both regions are required for optimal interaction with Ca(2+)-S100B. This work represents the first example of an S100B target that interacts with both the apo- and calcium-saturated forms of S100B.     

The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping

S100B is a homodimeric zinc-, copper-, and calcium-binding protein of the family of EF-hand S100 proteins. Zn²⁺ binding to S100B increases its affinity towards Ca²⁺ as well as towards target peptides and proteins. Cu²⁺ and Zn²⁺ bind presumably to the same site in S100B. We determined the structures of human Zn²⁺- and Ca²⁺-loaded S100B at pH 6.5, pH 9, and pH 10 by X-ray crystallography at 1.5, 1.4, and 1.65 Å resolution, respectively. Two Zn²⁺ ions are coordinated tetrahedrally at the dimer interface by His and Glu residues from both subunits. The crystal structures revealed that ligand swapping occurs for one of the four ligands in the Zn²⁺-binding sites. Whereas at pH 9, the Zn²⁺ ions are coordinated by His15, His25, His 85′, and His 90′, at pH 6.5 and pH 10, His90′ is replaced by Glu89′. The results document that the Zn²⁺-binding sites are flexible to accommodate other metal ions such as Cu²⁺. Moreover, we characterized the structural changes upon Zn²⁺ binding, which might lead to increased affinity towards Ca²⁺ as well as towards target proteins. We observed that in Zn²⁺-Ca²⁺-loaded S100B the C-termini of helix IV adopt a distinct conformation. Zn²⁺ binding induces a repositioning of residues Phe87 and Phe88, which are involved in target protein binding. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.     

Interactions of S100A2 and S100A6 with the tetratricopeptide repeat proteins, Hsp90/Hsp70-organizing protein and kinesin light chain

S100A2 and S100A6 interact with several target proteins in a Ca2+-regulated manner. However, the exact intracellular roles of the S100 proteins are unclear. In this study we identified Hsp70/Hsp90-organizing protein (Hop) and kinesin light chain (KLC) as novel targets of S100A2 and S100A6. Hop directly associates with Hsp70 and Hsp90 through the tetratricopeptide (TPR) domains and regulates Hop-Hsp70 and Hop-Hsp90 complex formation. We have found that S100A2 and S100A6 bind to the TPR domain of Hop, resulting in inhibition of the Hop-Hsp70 and Hop-Hsp90 interactions in vitro. Although endogenous Hsp70 and Hsp90 interact with Hop in resting Cos-7 cells, but not with S100A6, stimulation of these cells with ionomycin caused a Hop-S100A6 interaction, resulting in the dissociation of Hsp70 and Hsp90 from Hop. Similarly, glutathione S-transferase pulldown and co-immunoprecipitation experiments revealed that S100A6 binds to the TPR domain of KLC, resulting in inhibition of the KLC-c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1) interaction in vitro. The transiently expressed JIP-1 interacts with KLC in resting Cos-7 cells but not with S100A6. Stimulation of these cells with ionomycin also caused a KLC-S100A6 interaction, resulting in dissociation of JIP-1 from KLC. These results strongly suggest that the S100 proteins modulate Hsp70-Hop-Hsp90 multichaperone complex formation and KLC-cargo interaction via Ca2+-dependent S100 protein-TPR protein complex formation in vivo as well as in vitro. Moreover, we have shown that S100A2 and S100A6 interact with another TPR protein Tom70 and regulate the Tom70-ligand interaction in vitro. Thus, our findings suggest a new intracellular Ca2+-signaling pathway via S100 proteins-TPR motif interactions.     

Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: identification of putative drug binding sites on S100A1 protein

S100 proteins are a multigenic family of low-molecular-weight Ca(2+)-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca(2+)-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca(2+)-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1. These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family.     

Identification of regions responsible for the open conformation of S100A10 using chimaeric S100A11-S100A10 proteins

S100A11 is a dimeric EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2) and facilitate membrane vesiculation events. In contrast with other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an 'open' conformation that is very similar to S100A11 in its calcium-bound state. To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimaeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimaeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'disulfonic acid, dipotassium salt) fluorescence and phenyl-Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimaeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target binding site. In contrast, S100A11 chimaeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and, consequently, unobservable annexin A2 binding. The present study represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10.     

Mapping the zinc ligands of S100A2 by site-directed mutagenesis

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.     

S100-annexin complexes--structural insights

Annexins and S100 proteins represent two large, but distinct, calcium-binding protein families. Annexins are made up of a highly alpha-helical core domain that binds calcium ions, allowing them to interact with phospholipid membranes. Furthermore, some annexins, such as annexins A1 and A2, contain an N-terminal region that is expelled from the core domain on calcium binding. These events allow for the interaction of the annexin N-terminus with target proteins, such as S100. In addition, when an S100 protein binds calcium ions, it undergoes a structural reorientation of its helices, exposing a hydrophobic patch capable of interacting with its targets, including the N-terminal sequences of annexins. Structural studies of the complexes between members of these two families have revealed valuable details regarding the mechanisms of the interactions, including the binding surfaces and conformation of the annexin N-terminus. However, other S100-annexin interactions, such as those between S100A11 and annexin A6, or between dicalcin and annexins A1, A2 and A5, appear to be more complicated, involving the annexin core region, perhaps in concert with the N-terminus. The diversity of these interactions indicates that multiple forms of recognition exist between S100 proteins and annexins. S100-annexin interactions have been suggested to play a role in membrane fusion events by the bridging together of two annexin proteins, bound to phospholipid membranes, by an S100 protein. The structures and differential interactions of S100-annexin complexes may indicate that this process has several possible modes of protein-protein recognition.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: a novel mode for S100 protein target recognition

S100A6 is a member of the S100 subfamily of EF-hand Ca (2+) binding proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP or SIP), a subunit of an SCF-like E3 ubiquitin ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal S100A6 binding region in SIP was mapped to a 31-residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189-219) complex revealed that SIP(189-219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structure-based SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions.     

Three-dimensional solution structure of a unique S100 protein

S100A13 is a homodimeric protein that belongs to the S100 subfamily of EF-hand Ca2+-binding proteins. S100A13 exhibits unique physical and functional properties not observed in other members of the S100 family. S100A13 is crucial for the non-classical export of acidic fibroblast growth factors (FGFs-1), which lack signal peptide at their N-terminal end. In the present study, we report the three-dimensional solution structure of Ca2+-bound S100A13 using a variety of 3D NMR experiments. The structure of S100A13 is globular with four helices and an antiparallel beta-sheet in each subunit. The dimer interface is formed mainly by an antiparallel arrangement of helices H1, H1', H4, and H4'. Isothermal titration calorimetry (ITC) experiments show that S100A13 binds non-cooperatively to four calcium ions. Prominent differences exist between the three-dimensional structures of S100A13 and other S100 proteins. The hydrophobic pocket that largely contributes to protein-protein interactions in other S100 proteins is absent in S100A13. The structure of S100A13 is characterized by a large patch of negatively charged residues flanked by dense cationic clusters contributed largely by the positively charged residues located at the C-terminal end. Results of ITC experiments reveal that S100A13 lacking the C-terminal segment (residues 88-98) fails to bind FGF-1. The three-dimensional structure of S100A13 not only provides useful clues on its role in the non-classical export of signal peptide-less proteins such as FGF-1 but also paves the way for rational design of drugs against FGF-induced tumors.