Isabelle Durussel

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.

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.

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.

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.

S100A13 BIOCHEMICAL CHARACTERIZATION AND SUBCELLULAR LOCALIZATION IN DIFFERENT CELL LINES

S100 proteins became of major interest because of their divergent cell- and tissue-specific expression, their close association with a number of human diseases, and their importance for clinical diagnostics. Here, we report for the first time the purification and characterization of human recombinant S100A13. Flow dialysis revealed that the homodimeric S100A13 binds four Ca2+ in two sets of binding sites, both displaying positive cooperativity but of very different affinity. Fluorescence and difference spectrophotometry indicate that the Trp/Tyr signal changes are almost complete upon binding of Ca2+ to the two high affinity sites, which probably correspond to the C-terminal EF-hands in each subunit. The far-UV circular dichroic signal also changes upon binding of the first two Ca2+. So far, the tissue distribution of S100A13 has not been well characterized. Here, we show that S100A13 is widely expressed in various types of tissues with a high expression level in thyroid gland. Using specific antisera against S100A13, high protein expression was detected in follicle cells of thyroid, Leydig cells of testis, and specific cells of brain. In human smooth muscle cells, which co-express S100A2 in the nucleus and S100A1 in stress fibers, S100A13 shows a unique subcellular localization in the perinuclear area. These data suggest diverse functions for this protein in signal transduction.

S100A16, a Novel Calcium-binding Protein of the EF-hand Superfamily

S100A16 protein is a new and unique member of the EF-hand Ca2+-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 Alzheimers 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 Ca2+ 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 Ca2+ 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 Ca2+ stimulation.

Scherffelia dubia centrin exhibits a specific mechanism for Ca(2+)-controlled target binding.

Centrins are calcium binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. Herein, we report the molecular features and binding properties of the green alga Scherffelia dubia centrin (SdCen), a member of the Chlamydomonas reinhardtii centrin (CrCen) subfamily. The Ca(2+) binding capacity of SdCen and its isolated N- and C-terminal domains (N-SdCen and C-SdCen, respectively) was investigated using flow dialysis and isothermal titration calorimetry. In contrast with human centrin 1 and 2 (from the same subfamily), but like CrCen, SdCen exhibits three physiologically significant Ca(2+) binding sites, two in the N-terminal domain and one in the C-terminal domain. Mg(2+) ions could compete with Ca(2+) in one of the N-terminal sites. When Ca(2+) binds, the N-terminal domain becomes more stable and exposes a significant hydrophobic surface that binds hydrophobic fluorescent probes. The Ca(2+) binding properties and the metal ion-induced structural changes in the C-terminal domain are comparable to those of human centrins. We used isothermal titration calorimetry to quantify the binding of SdCen, N-SdCen, and C-SdCen to three types of natural target peptides, derived from the human XPC protein (P17-XPC), the human Sfi1 protein (R17-hSfi1), and the yeast Kar1 protein (P19-Kar1). The three peptides possess the complete (P17-XPC and R17-hSfi1) or partial (P19-Kar1) centrin binding motif (W(1)L(4)L(8)). The integral SdCen exhibits two binding sites for each target peptide, with distinct affinities for each site and each peptide. The high-affinity peptide binding site corresponds to the C-terminal domain of SdCen and displays binding constants and the poor Ca(2+) sensitivities similar to those observed for human centrins. The low-affinity site constituted by the N-terminal domain is active only in the presence of Ca(2+). The thermodynamic binding parameters suggest that the C-terminal domain of SdCen may be constitutively bound to a target, while the N-terminal domain could bind a target only after a Ca(2+) signal. SdCen is also able to interact with calmodulin binding peptides (W(1)F(5)V(8)F(14) motif) with a 1:1 stoichiometry, whereas the isolated N- and C-terminal domains have a much lower affinity. These data suggest particular molecular mechanisms used by SdCen (and probably by other algal centrins) to respond to cellular Ca(2+) signals.

Ion-binding properties of recombinant S100β and two derivatives with either an inactivated Ca2+ site II or a normalized Ca2+ site I

S100beta contains one unusual and one canonical Ca2+-binding motif. In this study, we measured Ca2+-binding and ensuing conformational changes of recombinant S100beta (rS100beta) and of two mutant forms in which either the canonical loop was inactivated (NoEF) or the unusual one replaced by a canonical one (Caloops). Caloops binds two Ca2+ per monomer with a 3-fold higher affinity than rS100beta; the affinity of NoEF was too low for accurate direct determination. All three proteins bind 3-4 Zn2+ per monomer. Tyrosine 17 fluorescence spectra showed a decrease of intensity upon binding of Ca2+ to the three proteins and an increase upon binding of Zn2+ to rS100beta and NoEF but not in Caloops. The fluorescence change as a function of the Ca2+ concentration yielded half-maximal changes ([Ca2+]0.5) at 1.7, 11.3 and 0.55 mM free Ca2+ for rS100beta NoEF and Caloops, respectively. Our data demonstrate that in S100beta alterations in one site can affect the Ca2+ binding properties of the other site.