Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA

Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.     

Calcium-dependent and -independent interactions of the S100 protein family

The S100 proteins comprise at least 25 members, forming the largest group of EF-hand signalling proteins in humans. Although the proteins are expressed in many tissues, each S100 protein has generally been shown to have a preference for expression in one particular tissue or cell type. Three-dimensional structures of several S100 family members have shown that the proteins assume a dimeric structure consisting of two EF-hand motifs per monomer. Calcium binding to these S100 proteins, with the exception of S100A10, results in an approx. 40 degrees alteration in the position of helix III, exposing a broad hydrophobic surface that enables the S100 proteins to interact with a variety of target proteins. More than 90 potential target proteins have been documented for the S100 proteins, including the cytoskeletal proteins tubulin, glial fibrillary acidic protein and F-actin, which have been identified mostly from in vitro experiments. In the last 5 years, efforts have concentrated on quantifying the protein interactions of the S100 proteins, identifying in vivo protein partners and understanding the molecular specificity for target protein interactions. Furthermore, the S100 proteins are the only EF-hand proteins that are known to form both homo- and hetero-dimers, and efforts are underway to determine the stabilities of these complexes and structural rationales for their formation and potential differences in their biological roles. This review highlights both the calcium-dependent and -independent interactions of the S100 proteins, with a focus on the structures of the complexes, differences and similarities in the strengths of the interactions, and preferences for homo- compared with hetero-dimeric S100 protein assembly.     

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.     

Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type.

A multigenic family of Ca2+-binding proteins of the EF-hand type known as S100 comprises 19 members that are differentially expressed in a large number of cell types. Members of this protein family have been implicated in the Ca2+-dependent (and, in some cases, Zn2+- or Cu2+-dependent) regulation of a variety of intracellular activities such as protein phosphorylation, enzyme activities, cell proliferation (including neoplastic transformation) and differentiation, the dynamics of cytoskeleton constituents, the structural organization of membranes, intracellular Ca2+ homeostasis, inflammation, and in protection from oxidative cell damage. Some S100 members are released or secreted into the extracellular space and exert trophic or toxic effects depending on their concentration, act as chemoattractants for leukocytes, modulate cell proliferation, or regulate macrophage activation. Structural data suggest that many S100 members exist within cells as dimers in which the two monomers are related by a two-fold axis of rotation and that Ca2+ binding induces in individual monomers the exposure of a binding surface with which S100 dimers are believed to interact with their target proteins. Thus, any S100 dimer is suggested to expose two binding surfaces on opposite sides, which renders homodimeric S100 proteins ideal for crossbridging two homologous or heterologous target proteins. Although in some cases different S100 proteins share their target proteins, in most cases a high degree of target specificity has been described, suggesting that individual S100 members might be implicated in the regulation of specific activities. On the other hand, the relatively large number of target proteins identified for a single S100 protein might depend on the specific role played by the individual regions that in an S100 molecule contribute to the formation of the binding surface. The pleiotropic roles played by S100 members, the identification of S100 target proteins, the analysis of functional correlates of S100-target protein interactions, and the elucidation of the three-dimensional structure of some S100 members have greatly increased the interest in S100 proteins and our knowledge of S100 protein biology in the last few years. S100 proteins probably are an example of calcium-modulated, regulatory proteins that intervene in the fine tuning of a relatively large number of specific intracellular and (in the case of some members) extracellular activities. Systems, including knock-out animal models, should be now used with the aim of defining the correspondence between the in vitro regulatory role(s) attributed to individual members of this protein family and the in vivo function(s) of each S100 protein.     

Characterization of the metastasis-associated protein,S100A4. Roles of calcium binding and dimerization in cellular localization and interaction with myosin

Elevated S100A4 protein expression is associated with metastatic tumor progression and appears to be a strong molecular marker for clinical prognosis. S100A4 is a calcium-binding protein that is known to form homodimers and interacts with several proteins in a calcium-dependent manner. Here we show that S100A4 localizes to lamellipodia structures in a migrating breast cancer-derived cell line and colocalizes with a known S100A4-interacting protein, myosin heavy chain IIA, at the leading edge. We demonstrate that S100A4 mutants that are defective in either their ability to dimerize or in calcium binding are unable to interact with myosin heavy chain IIA. An S100A4 mutant that is deficient for calcium binding retains the ability to form homodimers, suggesting that S100A4 can exist as calcium-free or calcium-bound dimers in vivo. However, a calcium-bound S100A4 monomer only interacts with another calcium-bound monomer and not with an S100A4 mutant that does not bind calcium. Interestingly, despite the calcium dependence for interaction with known protein partners, calcium binding is not necessary for localization to lamellipodia. Both wild type and a mutant that is deficient for calcium binding colocalize with known markers of actively forming leading edges of lamellipodia, Arp3 and neuronal Wiskott-Aldrich syndrome protein. These data suggest that S100A4 localizes to the leading edge in a calcium-independent manner, and identification of the proteins that are involved in localizing S100A4 to the lamellipodial structures may provide novel insight into the mechanism by which S100A4 regulates metastasis.     

Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity

S100 proteins comprise the largest family of calcium-binding proteins. Members of this family usually form homo- or heterodimers, which may associate to higher-order oligomers in a calcium-dependent manner. The heterodimers of S100A8 and S100A9 represent the major calcium-binding proteins in phagocytes. Both proteins regulate migration of these cells via modulation of tubulin polymerization. Calcium binding induces formation of (S100A8/S100A9)2 tetramers. The functional relevance of these higher-order oligomers of S100 proteins, however, is not yet clear. To investigate the importance of higher-order oligomerization for S100 proteins, we created a set of mutations within S100A9 (N69A, E78A, N69A+E78A) destroying the high-affinity C-terminal calcium-binding site (EF-hand II). Mutations in EF-hand II did not interfere with formation of the S100A8/S100A9 heterodimer as demonstrated by yeast two-hybrid experiments and pull-down assays. In contrast, mass spectrometric analysis and density gradient centrifugation revealed that calcium-induced association of (S100A8/S100A9)2 tetramers was strictly dependent on a functional EF-hand II in S100A9. Failure of tetramer formation was associated with a lack of functional activity of S100A8/S100A9 complexes in promoting the formation of microtubules. Thus, our data demonstrate that calcium-dependent formation of (S100A8/S100A9)2 tetramers is an essential prerequisite for biological function. This is the first report showing a functional relevance of calcium-induced higher-order oligomerization in the S100 family.     

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.     

CPT1A‐mediated succinylation of S100A10 increases human gastric cancer invasion

Gastric cancer (GC) is a malignancy of the lining of the stomach and is prone to distant metastasis, which involves a variety of complex molecules. The S100 proteins are a family of calcium‐binding cytosolic proteins that possess a wide range of intracellular and extracellular functions and play pivotal roles in the invasion and migration of tumour cells. Among these, S100A10 is known to be overexpressed in GC. Lysine succinylation, a recently identified form of protein post‐translational modification, is an important regulator of cellular processes. Here, we demonstrated that S100A10 was succinylated at lysine residue 47 (K47), and levels of succinylated S100A10 were increased in human GC. Moreover, K47 succinylation of S100A10 was stabilized by suppression of ubiquitylation and subsequent proteasomal degradation. Furthermore, carnitine palmitoyltransferase 1A (CPT1A) was found to function as a lysine succinyltransferase that interacts with S100A10. Succinylation of S100A10 is regulated by CPT1A, while desuccinylation is regulated by SIRT5. Overexpression of a succinylation mimetic mutant, K47E S100A10, increased cell invasion and migration. Taken together, this study reveals a novel mechanism of S100A10 accumulation mediated by succinylation in GC, which promotes GC progression and is regulated by the succinyltransferase CPT1A and SIRT5‐mediated desuccinylation.     

A logical sequence search for S100B target proteins

The EF-hand calcium-binding protein S100B has been shown to interact in vitro in a calcium-sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (alpha and beta), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled-coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of alpha and beta tubulin found at the alpha beta tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins.     

A novel S100 target conformation is revealed by the solution structure of the Ca2+-S100B-TRTK-12 complex

The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (K(d) <1 microm). In this work, the three-dimensional structure of the Ca(2+)-S100B-TRTK-12 complex has been determined by NMR spectroscopy. The structure reveals an extended, contiguous hydrophobic surface is formed on Ca(2+)-S100B for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp(7), and interacts with multiple hydrophobic contacts in helices III and IV of Ca(2+)-S100B. This interaction is strikingly different from the alpha-helical structures found for other S100 target peptides. By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV. This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions.     

Post-translational S-Nitrosylation Is an Endogenous Factor Fine Tuning the Properties of Human S100A1 Protein

S100A1 is a member of the Ca²⁺-binding S100 protein family. It is expressed in brain and heart tissue, where it plays a crucial role as a modulator of Ca²⁺ homeostasis, energy metabolism, neurotransmitter release, and contractile performance. Biological effects of S100A1 have been attributed to its direct interaction with a variety of target proteins. The (patho)physiological relevance of S100A1 makes it an important molecular target for future therapeutic intervention. S-Nitrosylation is a post-translational modification of proteins, which plays a role in cellular signal transduction under physiological and pathological conditions. In this study, we confirmed that S100A1 protein is endogenously modified by Cys⁸⁵ S-nitrosylation in PC12 cells, which are a well established model system for studying S100A1 function. We used isothermal calorimetry to show that S-nitrosylation facilitates the formation of Ca²⁺-loaded S100A1 at physiological ionic strength conditions. To establish the unique influence of the S-nitroso group, our study describes high resolution three-dimensional structures of human apo-S100A1 protein with the Cys⁸⁵ thiol group in reduced and S-nitrosylated states. Solution structures of the proteins are based on NMR data obtained at physiological ionic strength. Comparative analysis shows that S-nitrosylation fine tunes the overall architecture of S100A1 protein. Although the typical S100 protein intersubunit four-helix bundle is conserved upon S-nitrosylation, the conformation of S100A1 protein is reorganized at the sites most important for target recognition (i.e. the C-terminal helix and the linker connecting two EF-hand domains). In summary, this study discloses cysteine S-nitrosylation as a new factor responsible for increasing functional diversity of S100A1 and helps explain the role of S100A1 as a Ca²⁺ signal transmitter sensitive to NO/redox equilibrium within cells.     

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.     

Self-association of Calcium-binding Protein S100A4 and Metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.     

Sumoylation and Nuclear Translocation of S100A4 Regulate IL-1β-mediated Production of Matrix Metalloproteinase-13

S100A4, a member of the S100 family of proteins, plays an important role in matrix remodeling by up-regulating the expression of matrix metalloproteinases (MMPs). We have previously shown that S100A4 is overexpressed in diseased cartilage and that extracellular S100A4 stimulates MMP-13 production, a major type II collagen-degrading enzyme, via activation of receptor for advanced glycation end product signaling. In the present study, using human articular chondrocytes, we show that intracellular S100A4 translocated into the nucleus upon interleukin-1β (IL-1β) stimulation and translocation required post-translational modification of S100A4 by the sumo-1 protein. Two sumoylation sites were identified on the S100A4 molecule, Lys²² and Lys⁹⁶. Mutation of these lysine residues abolished the ability of S100A4 to be sumoylated and to translocate into the nucleus. Blocking of sumoylation and nuclear transport of S100A4 inhibited the IL-1β-induced production of MMP-13. Nuclear S100A4 was bound to the promoter region of MMP-13 in IL-1β-treated cells. Thus, we demonstrate a novel mechanism for sumoylated S100A4 as a regulator of expression of the MMP-13 gene.     

Crystal structure of metastasis-associated protein S100A4 in the active calcium-bound form

S100A4 (metastasin) is a member of the S100 family of calcium-binding proteins that is directly involved in tumorigenesis. Until recently, the only structural information available was the solution NMR structure of the inactive calcium-free form of the protein. Here we report the crystal structure of human S100A4 in the active calcium-bound state at 2.03 Å resolution that was solved by molecular replacement in the space group P6₅ with two molecules in the asymmetric unit from perfectly merohedrally twinned crystals. The Ca^2 +-bound S100A4 structure reveals a large conformational change in the three-dimensional structure of the dimeric S100A4 protein upon calcium binding. This calcium-dependent conformational change opens up a hydrophobic binding pocket that is capable of binding to target proteins such as annexin A2, the tumor-suppressor protein p53 and myosin IIA. The structure of the active form of S100A4 provides insight into its interactions with its binding partners and a better understanding of its role in metastasis.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

S100A16, a novel calcium-binding protein of the EF-hand superfamily

S100A16 protein is a new and unique member of the EF-hand Ca(2+)-binding proteins. S100 proteins are cell- and tissue-specific and are involved in many intra- and extracellular processes through interacting with specific target proteins. In the central nervous system S100 proteins are implicated in cell proliferation, differentiation, migration, and apoptosis as well as in cognition. S100 proteins became of major interest because of their close association with brain pathologies, for example depression or Alzheimer's disease. Here we report for the first time the purification and biochemical characterization of human and mouse recombinant S100A16 proteins. Flow dialysis revealed that both homodimeric S100A16 proteins bind two Ca(2+) ions with the C-terminal EF-hand of each subunit, the human protein exhibiting a 2-fold higher affinity. Trp fluorescence variations indicate conformational changes in the orthologous proteins upon Ca(2+) binding, whereas formation of a hydrophobic patch, implicated in target protein recognition, only occurs in the human S100A16 protein. In situ hybridization analysis and immunohistochemistry revealed a widespread distribution in the mouse brain. Furthermore, S100A16 expression was found to be astrocyte-specific. Finally, we investigated S100A16 intracellular localization in human glioblastoma cells. The protein was found to accumulate within nucleoli and to translocate to the cytoplasm in response to Ca(2+) stimulation.     

Structural basis of the Ca(2+)-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I

Background: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. Results: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction. Conclusions: By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.     

Recognition of the tumor suppressor protein p53 and other protein targets by the calcium-binding protein S100B

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.