The Zn2+ and Ca2+‐binding S100B and S100A1 proteins: beyond the myths

The S100 genes encode a conserved group of 21 vertebrate‐specific EF‐hand calcium‐binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium‐ and zinc‐binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn²⁺. The Ca²⁺ and Zn²⁺‐dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate‐specific proteins and propose a new model for stable interactions of S100B dimers with full‐length target proteins. A chaperone‐associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.     

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.     

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.     

S100A6 - New facts and features

S100A6 (calcyclin) is a 10.5 kDa Ca²⁺-binding protein that belongs to the S100 protein family. S100A6 contains two EF-hand motifs responsible for binding of Ca²⁺. It also binds Zn²⁺ through not yet identified structures. Binding of Ca²⁺ induces a conformational change in the S100A6 molecule which in consequence increases its overall hydrophobicity and allows for interaction with target proteins. S100A6 was found in different mammalian and avian (chicken) tissues. A high level of S100A6 is observed in epithelial cells, fibroblasts and in different kinds of cancer cells. The function of S100A6 is not clear at present, but it has been suggested that it may be involved in cell proliferation, cytoskeletal dynamics and tumorigenesis. Additionally, S100A6 might have some extracellular activities. This review presents new facts and features concerning the S100A6 protein.     

S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

Membrane Interactions of S100A12 (Calgranulin C)

S100A12 (Calgranulin C) is a small acidic calcium-binding peripheral membrane protein with two EF-hand structural motifs. It is expressed in macrophages and lymphocytes and highly up-regulated in several human inflammatory diseases. In pigs, S100A12 is abundant in the cytosol of granulocytes, where it is believed to be involved in signal modulation of inflammatory process. In this study, we investigated the interaction of the porcine S100A12 with phospholipid bilayers and the effect that ions (Ca²⁺, Zn²⁺ or both together) have in modifying protein-lipid interactions. More specifically, we intended to address issues such as: (1) is the protein-membrane interaction modulated by the presence of ions? (2) is the protein overall structure affected by the presence of the ions and membrane models simultaneously? (3) what are the specific conformational changes taking place when ions and membranes are both present? (4) does the protein have any kind of molecular preferences for a specific lipid component? To provide insight into membrane interactions and answer those questions, synchrotron radiation circular dichroism spectroscopy, fluorescence spectroscopy, and surface plasmon resonance were used. The use of these combined techniques demonstrated that this protein was capable of interacting both with lipids and with ions in solution, and enabled examination of changes that occur at different levels of structure organization. The presence of both Ca²⁺ and Zn²⁺ ions modify the binding, conformation and thermal stability of the protein in the presence of lipids. Hence, these studies examining molecular interactions of porcine S100A12 in solution complement the previously determined crystal structure information on this family of proteins, enhancing our understanding of its dynamics of interaction with membranes.     

Zinc ion binding to human brain calcium binding proteins. Calmodulin and S100b protein

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn²⁺ binding sites, with K_D ranging from 8.10^?5M to 3.10^?4M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. K_D = 10^?7 ? 10^?6M. We suggest that S100b protein should no longer be considered only as a “calcium binding protein” but also as a “zinc binding protein”, and that Zn²⁺ ions are involved in the functions of the S100 proteins.     

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     

RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins

The S100 protein family comprises at least 25 members which, with the exception of S100G, act as Ca2+-sensor proteins that participate in Ca2+ signal transduction by interacting with target proteins thereby modifying their activities. S100 proteins are expressed in vertebrates exclusively, display a cell-specific distribution, and regulate a large variety of intracellular activities. Some S100 proteins are released by a non-classical pathway and exert regulatory effects on several cell types. The receptor for advanced glycation end products (RAGE) has been shown to transduce extracellular effects of S100B, S100A4, S100A6, S100A11, S100A12, S100A13 and S100P. However, some S100 proteins can signal by engaging RAGE as well as non-RAGE receptors. Immune cells (i.e., monocytes/macrophages/microglia, neutrophils and lymphocytes), activated endothelial and vascular smooth muscle cells, neurons, astrocytes, chondrocytes and pancreatic tumor cells are the cell types reported to respond to certain S100 proteins via RAGE engagement. In general, relatively high concentrations of S100 proteins are required for activation of RAGE in responsive cells. S100B is unique in that it can engage RAGE in neurons at low and high concentrations with trophic and toxic effects, respectively, and S100A4 stimulates matrix metalloproteinase 13 release from chondrocytes at nanomolar doses in a RAGE-mediated manner. Oligomerization of S100 proteins under the non-reducing, high-Ca2+ conditions found extracellularly appears to play a relevant role in RAGE activation, and binding of at least S100A12 and S100B results in RAGE oligomerization. Thus, S100/RAGE interactions might have important consequences during development and in tissue homeostasis as well as in inflammatory, degenerative and tumor processes.     

Role of S100 proteins in health and disease

The S100 family of proteins contains 25 known members that share a high degree of sequence and structural similarity. However, only a limited number of family members have been characterized in depth, and the roles of other members are likely undervalued. Their importance should not be underestimated however, as S100 family members function to regulate a diverse array of cellular processes including proliferation, differentiation, inflammation, migration and/or invasion, apoptosis, Ca²⁺ homeostasis, and energy metabolism. Here we detail S100 target protein interactions that underpin the mechanistic basis to their function, and discuss potential intervention strategies targeting S100 proteins in both preclinical and clinical situations.     

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins.

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.     

Copper-mediated cross-linking of S100A4, but not of S100A2, results in proinflammatory effects in melanoma cells

The aim of this study was to investigate the response to and the physiological consequences of copper-mediated cross-linking of S100A2 and S100A4, two members of the S100 family of EF-hand calcium-binding proteins. As demonstrated by electrophoresis and mass spectrometry techniques S100A2 and S100A4 show formation of cross-links due to copper-mediated oxidation of cysteine residues. For S100A4, but not for S100A2, this results in both increased activation of NFκB and secretion of TNF-α in human A375 and, to a higher extent, in RAGE-transfected melanoma cells. The data suggest that a prooxidative tumor microenvironment enhances proinflammatory and prometastatic action of S100A4.     

Binding of transition metals to S100 proteins

The S100 proteins are a unique class of EF-hand Ca²⁺ binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn²⁺, Cu²⁺ and Mn²⁺ ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function.     

S100A11: Diverse Function and Pathology Corresponding to Different Target Proteins

S100A11, as a member of S100 protein family, while featuring the common identities as the other EF-hand Ca²⁺-binding family members, has its own individual characteristics. S100A11 is widely expressed in multiple tissues, and is located in cytoplasm, nucleus, and even cell periphery. S100A11 exists as a non-covalent homodimer with an antiparallel conformation. Ca²⁺ binding to S100A11 would trigger conformational changes which would expose the hydrophobic cleft of S100A11 and facilitate its interaction with target proteins. Since S100A11 appears to lack enzymatic activity, in this article, corresponding to a variety of its target proteins, we systematically describe the biological roles of S100A11 and its possible mechanism in the processes of inflammation, regulation of enzyme activity, and cell growth regulation. As a dual cell growth mediator, S100A11 acts as either a tumor suppressor or promoter in many different types of tumors and would play respective roles in influencing the proliferation of the cancer cells. We intend to illustrate the biological function of the S100 protein, and shed light on the further research, which will provide us with a better understanding of it.     

Subcellular distribution of S100 proteins in tumor cells and their relocation in response to calcium activation

 S100 proteins, a subgroup of the EF-hand Ca²⁺-binding protein family, regulate a variety of cellular processes via interaction with different target proteins. Several pathological disorders, including cancer, are linked to altered Ca²⁺ homeostasis and might involve the multifunctional S100 proteins, which are expressed in a cell- and tissue-specific manner. The present work demonstrates a distinct intracellular localization of S100A6, S100A4, and S100A2 in two tumor cell lines derived from metastatic epithelial breast adenocarcinoma (MDA-MB231) and cervical carcinoma (HeLa). Treatment of the cells by thapsigargin, the ionophore A23187, or cyclic ADP-ribose, to increase [Ca²⁺]_i via different pathways, led to relocation of S100A6 and S100A4 but only partially of the nuclear S100A2, as demonstrated by confocal laser scanning microscopy. These findings support the hypothesis that S100 proteins could play a crucial role in the regulation of Ca²⁺ homeostasis in cancer cells. Accepted: 3 March 1999     

The role of S100A4 protein in anticancer cytotoxicity: its presence is required on the surface of CD4+CD25+PGRPs+S100A4+ lymphocyte and undesirable on the surface of target cells

S100A4 is a Ca²⁺-binding protein that performs an important role in metastasis. It is also known for its antitumor functions. S100A4 is expressed by a specialized subset of CD⁴⁺CD²⁵⁺ lymphocytes and is present on those cell's membranes along with peptidoglycan recognition proteins (PGRPs). There, by interacting with major heat shock protein Hsp70, S100A4 plays an important cytotoxic role. The resulting stably formed complex of PGRPs, S100A4 and Hsp70 is required for the identification and binding between a lymphocyte and a target cell. Here, we investigated the S100A4 functions in CD⁴⁺CD²⁵⁺PGRPs⁺S100A4⁺ lymphocyte cytotoxicity against target cells. We demonstrated that those lymphocytes do not form a stable complex with the tumor target cells that themselves have S1004A on their surface. That observation can be explained by our finding that S100A4 precludes the formation of a stable complex between PGRPs, S100A4 (on the lymphocytes’ surface), and Hsp70 (on the target cells’ surface). The decrease in S100A4 level in CD⁴⁺CD²⁵⁺PGRPs⁺S100A4⁺ lymphocytes inhibits their cytotoxic activity, while the addition of S100A4 in the medium restores it. Thus, the resistance of target cells to CD⁴⁺CD²⁵⁺PGRPs⁺ S100A4⁺ lymphocyte cytotoxicity depends on their S100A4 expression level and can be countered by S100A4 antibodies.     

Inflammation-associated S100 proteins: new mechanisms that regulate function

This review focuses on new aspects of extracellular roles of the calgranulins. S100A8, S100A9 and S100A12 are constitutively expressed in neutrophils and induced in several cell types. The S100A8 and S100A9 genes are regulated by pro- and anti-inflammatory mediators and their functions may depend on cell type, mediators within a particular inflammatory milieu, receptors involved in their recognition and their post-translational modification. The S100A8 gene induction in macrophages is dependent on IL-10 and potentiated by immunosuppressive agents. S100A8 and S100A9 are oxidized by peroxide, hypochlorite and nitric oxide (NO). HOCl generates intra-chain sulfinamide bonds; stronger oxidation promotes cross-linked forms that are seen in human atheroma. S100A8 is >200-fold more sensitive to oxidative cross-linking than low-density lipoprotein and may reduce oxidative damage. S100A8 and S100A9 can be S-nitrosylated. S100A8–SNO suppresses mast cell activation and inflammation in the microcirculation and may act as an NO transporter to regulate vessel tone in inflammatory lesions. S100A12 activates mast cells and is a monocyte and mast cell chemoattractant; a G-protein-coupled mechanism may be involved. Structure–function studies are discussed in relation to conservation and divergence of functions in S100A8. S100A12 induces cytokines in mast cells, but not monocytes/macrophages. It forms complexes with Zn²⁺ and, by chelating Zn²⁺, S100A12 significantly inhibits MMPs. Zn²⁺ in S100A12 complexes co-localize with MMP-9 in foam cells in atheroma. In summary, S100A12 has pro-inflammatory properties that are likely to be stable in an oxidative environment, because it lacks Cys and Met residues. Conversely, S100A8 and S100A9 oxidation and S-nitrosylation may have important protective mechanisms in inflammation.     

Ca2+-Binding S100 Proteins in the Central Nervous System

A number of neurodegenerative disorders have been attributed to abberrations of intracellular Ca²⁺ homeostasis regulated by Ca²⁺-binding proteins. This chapter will focus on the S100B and S100A6 proteins, which are highly expressed in the central nervous system. Their protein structures, localizations, and association with brain pathology as well as their potential use in clinical diagnostics will be discussed.