Jos A. Cox

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

Cell-specific immuno-probes for the brain of normal and mutant Drosophila melanogaster

SummaryWe have screened antibodies for immunocytochemical staining in the optic lobes of the brain of Drosophila melanogaster. Seven polyclonal antisera and five monoclonal antibodies are described that selectively and reproducibly stain individual cells and/or produce characteristic staining patterns in the neuropile. Such antisera are useful for the cellular characterization of molecular and structural brain defects in visual mutants. In the wildtype visual system we can at present separately stain the following: the entire complement of columnar “ T 1” neurons; a small set of presumptive serotonergic neurons; some 3000 cells that contain and synthesize γ-amino butyric acid (GABA); and three groups of cells that bind antibodies to Ca2+-binding proteins. In addition, small groups of hitherto unknown tangential cells that send fine arborizations into specific strata of the medulla, and two patterns of characteristic layers in the visual neuropile have been identified by use of monoclonal antibodies generated following immunization of mice with homogenates of the brain of Drosophila melanogaster.

Brain S100A5 Is a Novel Calcium-, Zinc-, and Copper Ion-binding Protein of the EF-hand Superfamily

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca2+, Zn2+, and Cu2+. Flow dialysis revealed that the homodimeric S100A5 binds four Ca2+ ions with strong positive cooperativity and an affinity 20–100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn2+ ions and four Cu2+ ions per dimer. Cu2+ binding strongly impairs the binding of Ca2+; however, none of these ions change the α-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4′-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu2+, but not of Ca2+ or Zn2+, strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn2+ site is located at the opposite side of the EF-hands. The two Cu2+-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.

NADPH oxidase 5 (NOX5) interacts with and is regulated by calmodulin

Superoxide generation by NADPH oxidase 5 (NOX5) is regulated by Ca2+ through intramolecular activation of the C‐terminal catalytic domain by the EF‐hand‐containing N‐terminal regulatory domain. The C terminus contains a consensus calmodulin‐binding domain (CaMBD), which, however, is not the binding site of the N‐terminal regulatory domain. Here we show by pull down, cross‐linking, fluorimetry and by enzymatic assays, that calmodulin binds to this CaMBD in a Ca2+‐dependent manner, changes its conformation and increases the Ca2+ sensitivity of the N terminus‐regulated enzymatic activity. This mechanism represents an additional sophistication in the regulation of superoxide production by NOX5.

Cation- and peptide-binding properties of human centrin 2

Centrin and calmodulin (CaM) are closely related four‐EF‐hand Ca2+‐binding proteins. While CaM is monomeric, centrin 2 is dimeric and binds only two Ca2+ per dimer, likely to site IV in each monomer. Ca2+ binding to centrin 2 displays pronounced negative cooperativity and a [Ca2+]0.5 of 30 μM. As in CaM, Ca2+ binding leads to the exposure of a hydrophobic probe‐accessible patch on the surface of centrin 2. Provided Ca2+ is present, centrin 2 forms a 1:1 peptide:monomer complex with melittin with an affinity of 100 nM. The complex binds four instead of two Ca2+. Our data point to surprising differences in the mode of activation of these homologous proteins.

Purification and Cation Binding Properties of the Recombinant Human S100 Calcium-binding Protein A3, an EF-hand Motif Protein with High Affinity for Zinc

The calcium-binding protein S100A3 is an unusual member of the S100 family, characterized by its very high content of Cys. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified the recombinant human protein in Escherichia coli. The recombinant protein forms noncovalent homodimers, tetramers, and polymers in vitro with a subunit molecular weight of 11,712. The Zn2+-binding parameters of S100A3 were studied by equilibrium gel filtration and yielded a stoichiometry of four Zn2+ per monomer with a [Zn2+]0.5 of 11 μM and a Hill coefficient of 1.4 at physiological ionic strength. The affinity for Ca2+ is too low to be determined by direct methods (KCa > 10 mM). Ca2+- and Zn2+-binding can be followed by optical methods: the Trp-45 fluorescence is high in the metal-free form and addition of Zn2+ and Ca2+, but not of Mg2+, leads to a 4-fold quenching. Ca2+ and Zn2+ promote also quite similar conformational changes in the Tyr and Trp environment as monitored by difference spectrophotometry. Fluorescence titrations with Zn2+ confirmed that there is one set of high affinity binding sites with a [Zn2+]0.5 of 8 μM and a Hill coefficient of 1.3. Binding of Zn2+ to a second set of low affinity sites induces protein precipitation. Fluorescence titrations with Ca2+ confirmed the very low affinity of S100A3 for this ion with a [Ca2+]0.5 of 30 mM and slight negative cooperativity. Mg2+ has no effect on this binding curve. Of the 10 Cys residues in S100A3, 5 only are free thiols, and accessible to 5,5′-dithiobis(2-nitrobenzoic acid); they display a high reactivity in the metal-free and Ca2+ form, but a 20-fold lowered reactivity in the Zn2+ form of S100A3. Ca2+-binding promotes the formation of a solvent-accessible hydrophobic surface as monitored by the 60-fold fluorescence increase of 2-p-toluidinylnaphthalene-6-sulfonate, whereas Zn2+ has no noticeable influence. Our data indicate that Ca2+ and Zn2+ do not bind to the same sites and that under physiological conditions S100A3 is a Zn2+-binding rather than a Ca2+-binding protein; nevertheless, very specific conformational changes are introduced by either Ca2+ or Zn2+. Since no Zn2+-binding motif of known structure was identified in the primary sequence of S100A3, the results are suggestive for a novel Zn2+-binding motif.

Biochemical Characterization of the Penta-EF-hand Protein Grancalcin and Identification of L-plastin as a Binding Partner

Grancalcin is a recently described Ca2+-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca2+ although it has an unusual Ca2+ coordination loop and one that does not bind Ca2+ but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca2+ load. Flow dialysis showed that recombinant grancalcin binds two Ca2+ per subunit with positive cooperativity and moderate affinity ([Ca2+]0.5 of 25 and 83 μm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca2+-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca2+ and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca2+-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa.

Sarcoplasmic calcium-binding protein

Sarcoplasmic calcium-binding proteins (SCPs) are members of the EF-hand calcium-binding protein family which are characterized by the presence of helix-loop-helix motifs in their amino acid sequence. SCPs have an M(r) of approximately 20,000, a pI of approximately 5 and interact with two to three calcium ions (Ca2+) with a KD of 10(-7) to 10(-8) M. Mg2+ ions antagonize Ca2+ ion binding in a complex manner so that these proteins are exquisitely fine-tuned to interfere with the Ca2+ signal. SCPs apparently fulfil no specific activatory function. They exhibit strong polymorphism, show a marked homology to coelenterate photoproteins (aequorin, luciferin) and have been found only in invertebrates, predominantly in muscle and neurons. In mollusks, SCPs are distributed in a tissue-specific manner, with immunoreactivity to SCP I-like isoforms localized in electrically silent neurons colocalized with serotonin, and immunoreactivity to SCP II-like isoforms exclusively present in muscle.

EF‐hand motifs in inositol phospholipid‐specific phospholipase C

Computer sequence analysis with a linear weight matrix revealed the presence of one canonical EF‐hand motif in the δ isoform of inositol phospholipid‐specific phospholipase C and one ancestral EF‐hand in the γ form.

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of α-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in α-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2–3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.