Marie-Claude Kilhoffer

Calcium signalling in Bacillus subtilis

Few systematic studies have been devoted to investigating the role of Ca2+ as an intracellular messenger in prokaryotes. Here we report an investigation on the potential involvement of Ca2+ in signalling in Bacillus subtilis, a Gram-positive bacterium. Using aequorin, it is shown that B. subtilis cells tightly regulate intracellular Ca2+ levels. This homeostasis can be changed by an external stimulus such as hydrogen peroxide, pointing to a relationship between oxidative stress and Ca2+ signalling. Also, B. subtilis growth appears to be intimately linked to the presence of Ca2+, as normal growth can be immediately restored by adding Ca2+ to an almost non-growing culture in EGTA containing Luria broth medium. Addition of Fe2+ or Mn2+ also restores growth, but with 5-6 h delay, whereas Mg2+ did not have any effect. In addition, the expression of alkyl hydroperoxide reductase C (AhpC), which is strongly enhanced in bacteria grown in the presence of EGTA, also appears to be regulated by Ca2+. Finally, using 45Ca2+ overlay on membrane electrotransferred two-dimensional gels of B. subtilis, four putative Ca2+ binding proteins were found, including AhpC. Our results provide strong evidence for a regulatory role for Ca2+ in bacterial cells.

Terbium as luminescent probe of calmodulin calcium-binding sites: Domains I and II contain the high-affinity sites

Terbium has been shown to be a useful tool in the study of protein calcium binding sites [l]. Due to its ionic radius, its coordination number between 6 and 8 and its strong propensity for oxygen donor groups, this trivalent ion can replace calcium in many biological systems [2]. In addition, terbium has the convenient property of being highly luminescent when it binds to a protein close to an aromatic residue, as a result of a dipole-quadrupole energy-transfer process. By contrast the free ion is only poorly luminescent. Calmodulin, the ubiquitous and multifunctional calciumdependent regulator [3-5lexhibits 4 calcium binding sites among which sites III and IV contain one tyrosine residue each (tyrosine 99 in site III and tyrosine 138 in site IV [6]). No other tyrosyl residue is present in the molecule. Upon calcium binding, calmodulin undergoes a conformational transition which is necessary for its biological activity [7-91. Here, calmodulin conformational changes were induced by Ca*+ and Tb* and studied by monitoring tyrosine fluorescence, since calmodulin contains no tryptophan residues. Two calcium binding sites were shown to be involved in tyrosine fluorescence changes. A study of terbium emission indicates these high affinity sites to be sites I and II, in contrast to troponin C where the high affinity sites are sites III and IV.

Terbium binding to octopus calmodulin provides the complete sequence of ion binding

Calmodulin, the ubiquitous and multifunctional calcium-dependent regulator [ 1,2], exhibits 4 calciumbinding sites. For each of these sites, Ca*+ compete with Mg*” and K’ (J. Haiech, C. B. Klee, J. G. D., in preparation). Binding of Ca” to calmodulin induces a conformational change which was observed by different techniques, CD and UV difference spectra 131, NMR [4] or tyrosine fluorescence (151, M. C. K., J. G. D., D. G., in preparation}. Ca*-loaded calmodulin is able to interact with and activate a number of enzymes (reviewed [ 1,2]). It is still a matter of speculation whether all enzymes are activated by calmodulin when loaded with 4 Ca2+ or whether calmodulin can activate some enzymes after binding 2 or 3 Ca2+ only. It is therefore important to determine the sequence of ion binding to the 4 domains recognized in the primary structure of the protein [6]. Homologous calmodulins in spite of a very low rate of evolution [7] exhibit amino acid substitutions that may be useful in this respect. For instance, mammalian calmodulins contain two tyrosyl residues at positions 99 (domain HI) and 138 (domain IV) [6]. We have recently shown that ‘high affinity’ sites are sites I and II, since Tb3+ luminescence increases only after binding of 2 Tbs+ equiv~ents. The 2 first Tb3+ therefore bind to sites that lack tyrosine, namely domains I

A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes☆

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four EF hand type Ca(2+) binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca(2+) ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca(n) (n=0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Distribution of separations between groups in an engineered calmodulin.

An engineered calmodulin (VU-9-CaM) has been prepared in which a tryptophan group is present at position 99 and a tyrosine at position 138. The tyrosine was converted to nitrotyrosine. Timedomain dynamic fluorescence measurements were made of energy transfer from the tryptophan donor to the nitrotyrosine acceptor. These were analyzed to yield the parameters characterizing the distribution of separations between the two groups, which are located on Ca2+-binding domains III and IV. Their mean separation is in reasonable agreement with the crystallographic value.

The effect of plasma gelsolin on actin filaments Ca2+-dependency of the capping and severing activities

The Ca2+-dependency of plasma gelsolin capping and severing properties was investigated using viscometry, fluorescence of N-(1-pyrenyliodoacetamide)-labelled actin and Triton X-100 cell models. The two properties of plasma gelsolin appear to be expressed differently, severing being fully Ca2+-dependent, whereas capping seems to take place, even in the absence of Ca2+, although less efficiently.