Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations

S100A8 and S100A9 are two proinflammatory molecules belonging to the S100 family of calcium-binding proteins. Common to all S100 proteins S100A8 and S100A9 form non-covalently associated complexes which have been shown to exhibit different functional properties. Besides dimerization, recent research is focused on the importance of higher oligomeric structures of S100 proteins induced by bivalent cations. While S100A8/S100A9-heterodimers are formed in the absence of calcium, tetramerization is strictly calcium-dependent. Heterodimer formation is not a simple process and our biophysical analyses (FRET, ESI-MS) demonstrate that simply mixing both subunits is not sufficient to induce complex formation. Steps of denaturation/renaturation are necessary for the recombinant complex to show identical biophysical properties as S100A8/S100A9 obtained from granulocytes. In addition to calcium both proteins are able to bind zinc with high affinity. Here we demonstrate for the first time by different biophysical methods (MALDI-MS, ESI-MS, fluorescence spectroscopy) that zinc-binding, like calcium, induces (S100A8/S100A9)(2)-tetramers. Using mass spectrometric investigations we demonstrate that zinc triggers the formation of (S100A8/S100A9)(2)-tetramers by zinc-specific binding sites rather than by interactions with calcium-specific EF-hands. The zinc-induced tetramer is structurally very similar to the calcium-induced tetramer. Thus, like calcium, zinc acts as a regulatory factor in S100A8/S100A9-dependent signaling pathways.     

Molecular basis of the complex formation between the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14)

S100 proteins form characteristic homo- and/or heterodimers that play a role in calcium-mediated signaling. We characterized the formation of the human S100A8/S100A9 heterodimer using the yeast two-hybrid system. Employing site-directed mutagenesis we found that distinct hydrophobic amino acids of helix I/I' are located at a crucial site of the S100A8/S100A9 dimer interface, whereas conserved residues within helix IV/IV' are not important for heterodimerization. Furthermore, amino acids Y16 and F68 prevent homodimerization of human S100A8. These data demonstrate for the first time the functional relevance of distinct hydrophobic amino acids for human S100A8/S100A9 complex formation in vivo.     

S100A12 is expressed exclusively by granulocytes and acts independently from MRP8 and MRP14.

Changes in cytosolic calcium concentrations regulate a wide variety of cellular processes, and calcium-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control. S100A12, a recently described member of the S100 protein family, has been shown to be coexpressed in granulocytes and monocytes together with two other S100 proteins, MRP8 (S100A8) and MRP14 (S100A9), and a functional relationship between these three S100 proteins has been suggested. Using Western blotting, calcium overlays, intracellular flow cytometry, and cytospin preparations, we demonstrate that S100A12 expression in leukocytes is specifically restricted to granulocytes and that S100A12 represents one of the major calcium-binding proteins in these cells. S100A12, MRP8, and MRP14 translocate simultaneously from the cytosol to cytoskeletal and membrane structures in a calcium-dependent manner. However, no evidence for direct protein-protein interactions of S100A12 with either MRP8 or MRP14 or the heterodimer was found by chemical cross-linking, density gradient centrifugation, mass spectrometric measurements, or yeast two hybrid detection. Thus, S100A12 acts individually during calcium-dependent signaling, independent of MRP8, MRP14, and the heterodimer MRP8/MRP14. This granulocyte-specific signal transduction pathway may offer attractive targets for therapeutic intervention with exaggerated granulocyte activity in pathological states.     

Solution structure and dynamics of human S100A14

Human S100A14 is a member of the EF-hand calcium-binding protein family that has only recently been described in terms of its functional and pathological properties. The protein is overexpressed in a variety of tumor cells and it has been shown to trigger receptor for advanced glycation end products (RAGE)-dependent signaling in cell cultures. The solution structure of homodimeric S100A14 in the apo state has been solved at physiological temperature. It is shown that the protein does not bind calcium(II) ions and exhibits a “semi-open” conformation that thus represents the physiological structure of the S100A14. The lack of two ligands in the canonical EF-hand calcium(II)-binding site explains the negligible affinity for calcium(II) in solution, and the exposed cysteines and histidine account for the observed precipitation in the presence of zinc(II) or copper(II) ions.     

Zinc binding reverses the calcium-induced arachidonic acid-binding capacity of the S100A8/A9 protein complex

Analysis of the calcium-induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 calciums per EF-hand. The AA binding capacity was not induced by the binding of other bivalent cations, such as Zn2+, Cu2+, and Mg2+, to the protein complex. In contrast, the binding of AA was prevented by the addition of either Zn2+ or Cu2+ in the presence of calcium, whereas Mg2+ failed to abrogate the AA binding capacity. The inhibitory effect was not due to blocking the formation of S100A8/A9 as demonstrated by a protein-protein interaction assay. Fluorescence measurements gave evidence that both Zn2+ and Cu2+ induce different conformational changes thereby affecting the calcium-induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

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.     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.     

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.     

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.     

S100A7, S100A10, and S100A11 are transglutaminase substrates

S100 proteins are a family of 10-14 kDa EF-hand-containing calcium binding proteins that function to transmit calcium-dependent cell regulatory signals. S100 proteins have no intrinsic enzyme activity but bind in a calcium-dependent manner to target proteins to modulate target protein function. Transglutaminases are enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine bonds between protein-bound glutamine and lysine residues. In the present study we show that transglutaminase-dependent covalent modification is a property shared by several S100 proteins and that both type I and type II transglutaminases can modify S100 proteins. We further show that the reactive regions are at the solvent-exposed amino- and carboxyl-terminal ends of the protein, regions that specify S100 protein function. We suggest that transglutaminase-dependent modification is a general mechanism designed to regulate S100 protein function.     

Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.     

Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation

S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two alpha subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca(2+)-titration curves of Trp90 fluorescence for both the native protein and its Glu32 --> Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 --> Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K(1) =4 +/- 2 x 10(3) m(-1) (C-loops) and K(2) approximately 10(2) m(-1) (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid - a fluorescent probe of protein-surface hydrophobicity. S-Glutathionylation of the single cysteine residue (85) of the alpha subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous - four orders of magnitude - increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding to DeltaDeltaG = -6 +/- 1 kcal.mol(-1). A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.     

Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.     

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.     

Intrinsic disorder in S100 proteins

Although the members of the largest subfamily of the EF-hand proteins, S100 proteins, are evolutionarily young, their functional diversity is extremely broad, partly due to their ability to adapt to various targets. This feature is a hallmark of intrinsically disordered proteins (IDPs), but none of the S100 proteins are recognized as IDPs. S100 are predicted to be enriched in intrinsic disorder, with 62% of them being predicted to be disordered by at least one of the predictors: 31% are recognized as 'molten globules' and 15% are shown to be in extended disordered form. The disorder level of predicted disordered S100 regions is conserved compared to that of more structured regions. The central disordered stretch corresponds to the major part of pseudo EF-hand loop, helix II, hinge region, and an initial part of helix III. It contains about half of known sites of enzymatic post-translational modifications (PTMs), confirming that this region can be flexible in vivo. Most of the internal residues missing in tertiary structures belong to the hinge. Both hinge and pseudo EF-hand loop correspond to the local maxima of the PONDR? VSL2 score and are shown to be evolutionary hotspots, leading to gain of new functional properties. The action of PTMs is shown to be destabilizing, in contrast with the effect of metal-binding or S100 dimerization. Formation of the S100 heterodimers relies on the interplay between the structural rigidity of one of the S100 monomers and the flexibility of another monomer. The ordered regions dominate in the S100 homodimerization sites. Target-binding sites generally consist of distant regions, drastically differing in their disorder level. The disordered region comprising most of the hinge and the N-terminal half of helix III is virtually not involved into dimerization, being intended solely for target recognition. The structural flexibility of this region is essential for recognition of diverse target proteins. At least 86% of multiple interactions of S100 proteins with binding partners are attributed to the S100 proteins predicted to be disordered. Overall, the intrinsic disorder is inherent to many S100 proteins and is vital for activity and functional diversity of the family.     

S100A4与肿瘤细胞增殖及凋亡的研究进展

转移相关蛋白S100A4属于S100钙离子结合蛋白超家族,有着共同的EF手型功能区来介导其活性。S100A4在众多肿瘤生物学行为中起着调节作用。而且,S100A4在不同类型的肿瘤患者扮演着判断预后的角色。目前研究认为其与肿瘤细胞运动、增殖、刺激血管生成及基质重建有关系。本文就S100A4生物学特性及其对肿瘤细胞增殖、凋亡中的作用和可能机制作一综述。     

Analysis of the structure of human apo-S100B at low temperature indicates a unimodal conformational distribution is adopted by calcium-free S100 proteins

S100B is one of the best-characterized members of the calcium-signaling S100 protein family. Most S100 proteins are dimeric, with each monomer containing two EF-hand calcium-binding sites (EF1, EF2). S100B and other S100 proteins respond to calcium increases in the cell by coordinating calcium and undergoing a conformational change that allows them to interact with a variety of cellular targets. Although several three dimensional structures of S100 proteins are available in the calcium-free (apo-) state it has been observed that these structures appear to adopt a wide range of conformations in the EF2 site with respect to the positioning of helix III, the helix that undergoes the most dramatic calcium-induced conformational change. In this work, we have determined the structure of human apo-S100B at 10 degrees C to examine whether temperature might be responsible for these structural differences. Further, we have used this data, and other available apo-S100 structures, to show that despite the range of interhelical angles adopted in the apo-S100 structures, normal Gaussian distributions about the mean angles found in the structure of human apo-S100B are observed. This finding, only obvious from the analysis of all available apo-S100 proteins, provides direct structural evidence that helix III is a loosely packed helix. This is likely a necessary functional property of the S100 proteins that facilitates the calcium-induced conformational change of helix III. In contrast, the calcium-bound structures of the S100 proteins show significantly smaller variability in the interhelical angles. This shows that calcium binding to the S100 proteins causes not only a conformational change but results in a tighter distribution of helices within the EF2 calcium binding site required for target protein interactions.     

Effects of S100A9 in a rat model of asthma and in isolated tracheal spirals

S100A9 is a member of the S100 family of proteins that contain two EF-hand calcium-binding motifs. We previously reported that S100A9 was differentially expressed during the early airway response phase of asthma and can be regulated by acupuncture. To understand the possible role of S100A9 in asthma, the effects of the S100A9 were investigated in a rat model of asthma and in isolated tracheal spirals. The pulmonary function and isometric tension were measured after the administration of purified recombinant S100A9. The results of in vivo experiments showed that S100A9 (0.1 μg/kg) significantly decreased the pulmonary resistance and increased the dynamic compliance. The in vitro experimental results showed that S100A9 (100, 200, 400, or 800 ng/ml, final concentrations) significantly reduced the isometric tension of isolated tracheal spirals. These results suggest that S100A9 elicits dose-dependent anti-asthmatic effects and may provide further insight into the treatment of asthma.     

The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting alpha-helices can determine specific association of two EF-hand proteins

The EF-hand proteins S100A8 and S100A9 are important calcium signalling proteins that are involved in wound healing and provide clinically relevant markers of inflammatory processes, such as rheumatoid arthritis and inflammatory bowel disease. Both can form homodimers via distinct modes of association, probably of lesser stability in the case of S100A9, whereas in the presence of calcium S100A8 and S100A9 associate to calprotectin, the physiologically active heterooligomer. Here we describe the crystal structure of the (S100A8/S100A9)(2) heterotetramer at 1.8 A resolution. Its quaternary structure illustrates how specific heteroassociation is energetically driven by a more extensive burial of solvent accessible surface areas in both proteins, most pronounced for S100A9, thus leading to a dimer of heterodimers. A major contribution to tetramer association is made by the canonical calcium binding loops in the C-terminal halves of the two proteins. The mode of heterodimerisation in calprotectin more closely resembles the subunit association previously observed in the S100A8 homodimer and provides trans stabilisation for S100A9, which manifests itself in a significantly elongated C-terminal alpha-helix in the latter. As a consequence, two different putative zinc binding sites emerge at the S100A8/S100A9 subunit interface. One of these corresponds to a high affinity arrangement of three His residues and one Asp side-chain, which is unique to the heterotetramer. This structural feature explains the well known Zn(2+) binding activity of calprotectin, whose overexpression can cause strong dysregulation of zinc homeostasis with severe clinical symptoms.