Co-operation of domain-binding and calcium-binding sites in the activation of gelsolin Emeline Lagarrigue 1 , Sutherland K. Maciver 2 , Abdellatif Fattoum 3 , Yves Benyamin 1 and Claude Roustan 1 1 UMR 5539 (CNRS) Laboratoire de motilite ´ cellulaire (Ecole Pratique des Hautes Etudes), Universite ´ de Montpellier 2, France; 2 Genes and Development Group, Department of Biomedical Sciences, University of Edinburgh, Scotland, UK; 3 Centre de Recherches de Biochimie Macromole ´ culaire, UPR 1086 (CNRS), Montpellier, France Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a Ôhelical-latchÕ at the C-terminus of gelsolin’s domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (K d ¼15 l M ). We also report a calcium dependent binding site for the C-terminus (G4–6) within G2 and delimit this further to a specific region formed by residues 203–225 and 159–193. It is known that the acti- vation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium-dependent con- formational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4–6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physi- cal coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule. Keywords: actin; gelsolin; cytoskeleton; severing; calcium activation. Gelsolin is a calcium-activated actin filament severing and capping protein found in many tissues and in the plasma of vertebrates (for a review, see [1]). It belongs to a wider group of actin-binding proteins that share a number of repeated domains; six in the case of gelsolin itself, adseverin and villin, and three in capG, fragmin and severin (for a review, see [2]). The binding of calcium to gelsolin and to actin bound gelsolin is complex. Free gelsolin binds at least six calcium ions. These sites, coordinated solely by gelsolin have been termed type II [3]. The affinity of type II sites varies greatly. High affinity calcium sites (K d  1 l M ) have been identified [4–6] and two of these have been localized within G4–6 [7]. A body of evidence suggests that calcium binding by G4–6 affords calcium-sensitivity to the whole gelsolin molecule [8,9]. Sites have been identified by biochemical means within G4-5 (K d  2 l M ) and G5–6 (K d  0.2 l M ) [9], and crystallographic studies (S. Kolappan, J. Gooch, A. Weeds & P. McLaughlin, Wellcome Centre for Cell Biology, University of Edinburgh, UK, personal commu- nication, [3]) have shown that calcium ions are bound by both G5 and G6 (sites IIG5 and IIG6 [3]. Low affinity calcium-binding sites (K d  1m M ) have also been detected [10,11]. A site has been inferred to lie within G2-3 by proteolysis susceptibility and molecular radius changes [12], and this site has been narrowed further to G2 (IIG2) and tentatively suggested to have a dissociation constant of  32 l M [11]. Additionally, a calcium ion (IG1) is ÔtrappedÕ between actin and G1 [13,14], and by actin and G4 (IG4) [3,15], these calcium sites, coordinated by both gelsolin and actin have been termed type I sites [3]. In the absence of calcium, gelsolin cannot bind actin as its three [5,16] identified actin binding sites residing in G1, G2 and G4 [7,16,17] are not accessible. In the presence of calcium, gelsolin becomes activated by the unfolding of the whole molecule so that the F-actin binding region in G2 is exposed allowing the molecule to make the initial contact with the actin filament. Whereas gelsolin is opened by 0.1–1 l M calcium [12,18,19], the ternary actin: gelsolin complex is only stable at calcium concentrations exceeding Correspondence to C. Roustan, UMR 5539(CNRS) UM2 CC107, Place E. Bataillon 34095 Montpellier Cedex 5, France. Fax: + 33 0467144927, E-mail: roustanc@crit.univ-montp2.fr Abbreviations: G1-6, The six repeated domains of gelsolin; FITC, fluorescein 5-isothiocyanate; 1,5-I-AEDANS, N-iodoacetyl-N¢-(sulfo- 1-naphthyl)-ethylenediamine; BACNHS, biotinamidocaproate N-hydroxyl-succinimide ester; G-actin, monomeric actin; F-actin, filamentous actin. Note: we have adopted the labeling system introduced by Choe et al. [Choe, H., Burtnick, L.D., Mejillano, M., Yin, H.L., Robinson, R.C. & Choe, S. (2002) J. Mol. Evol. 324, 691–702.] for the various Calcium- binding sites so that IG1 is the type I binding site within G2 and IIG6 is the type II binding site within G6. Type I binding sites are coordinated by gelsolin and actin whereas type II sites are coordinated solely by gelsolin residues. Note: webpages are available at http://www.dbs.University-montp2.fr/ umr5539/, http://www.ephe.University-montp2.fr and http://www.bms.ed.ac.uk/research/others/smaciver/index.htm (Received 20 December 2002, revised 10 March 2003, accepted 26 March 2003) Eur. J. Biochem. 270, 2236–2243 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03591.x 30 l M [19], and the most rapid severing rate of actin filaments occurs at 300 l M [20]. Accumulating evidence [3,12,18,21] suggests the following mechanism for the activation of gelsolin by calcium. Low calcium concentra- tions are proposed [18,21,22] to ÔunlatchÕ the connection between G2 and G6, but higher concentrations are required to break salt bridges between other domains until the gelsolin is fully ÔopenÕ, then additional calcium ions are requiredtobindactintoG1andtoG4forseveringand capping. The details of the latch helix structure in the presence of calcium are not clear as there is no density for this region in the available crystallographic solutions [3,15], so we cannot tell if calcium ions are bound directly to the latch helix to inhibit its binding to G2. However, this seems unlikely as is has been shown that gelsolin without the C-terminal helix alters neither the number nor the affinity of gelsolin’s calcium binding sites [18]. Here, we confirm that G2 shows calcium-dependent conformational changes and that peptides derived from it show calcium-dependent binding to G4–6. Therefore the calcium-dependence shown by G2 may have a role in the multistep mechanism of calcium-activation of gelsolin prior to binding actin. Methods Proteins and peptides Human gelsolin domain 2 (G2) and domains 4–6 (G4–6) were produced in Escherichia coli BL21(pLysS) and BL21(de3), respectively, using the pMW172 vector [23], following induction of expression with isopropyl thio b- D - galactoside. G2 (residues 151–266 of human serum gelsolin) was purified from the soluble fraction of the bacteria [24], and G4–6 (residues 407–755) [25] was purified from inclusion bodies [26]. Gelsolin G4–6 domain was selectively cleaved by trypsin. The domain (225 lgÆmL )1 ) was incubated in 0.1 M Tris HCl pH 7.5 supplemented with either 1 m M CaCl 2 or 1 m M EGTA in the presence of trypsin (9 lg/mL). The proteolysis was stopped by addition of an antiprotease mixture (Complete) (1 : 25 mass/vol.) purchased from Roche SA (Mannheim, Germany). To identify the cleavage products, the digest was labeled at cysteine residues by treatment with 1,5-I-AEDANS (10 molar excess) [27] for 4 h at room temperature, in the presence of 1% SDS. The labeling reaction was stopped by addition of 0.1 M b-mercapto- ethanol. The digest was then analyzed by SDS/PAGE. Antibodies directed towards gelsolin G4–6 domain were elicited in rabbits according to [28]. Anti-IgG antibodies labeled with alkaline phosphatase were purchased from Sigma (Dorset, UK). Synthetic peptides derived from gelsolin sequences 159– 193 and 203–225 [17] were prepared on solid phase support using a 9050 Milligen PepSynthesizer (Millipore) according to the Fmoc/tBu system. The crude peptides were de-protected and thoroughly purified by preparative reverse-phase HPLC. The purified peptides were shown to be homogenous by analytical HPLC. Electrospray mass spectra, carried out in the positive ion mode using a Trio 2000 VG Biotech Mass spectrometer (Altrincham), were in line with the expected structures. Gelsolin G2 and G4–6 domains were labeled by FITC as described elsewhere [29]. Biotinylation of G2 domain by BACNHS was performed as reported previously [30]. Excess reagent was eliminated by chromatography on a PD10 column (Pharmacia) in 0.1 M NaHCO 3 buffer pH 8.6. Immunological techniques The ELISA technique [31], described previously in detail [32] was used to monitor interaction of ligands with synthetic peptides or G4–6 domain. G4–6 domain (1 lgÆmL )1 ) or synthetic peptides (1 lgÆmL )1 )in50m M NaHCO 3 /Na 2 CO 3 , pH 9.5, were immobilized on plastic microtiter wells. The plate was then saturated with 0.5% gelatin/3% gelatin hydrolysate in 140 m M NaCl, 0.05% tween 20, 10 m M Phosphate buffer, pH 7.4. Experiments with coated fragments were performed in 0.1 M KCl, 20 m M Tris pH 7.2. Binding was monitored at 405 nm using alkaline phosphatase-labeled anti-IgG antibodies (dilution 1/1000) or alkaline phosphatase labeled streptavidin (dilu- tion 1/1000). Control assays were carried out in wells saturated with gelatin and gelatin hydrolysate used alone. Each assay was conducted in triplicate and the mean value plotted after subtraction of nonspecific absorption. The binding parameters (apparent dissociation constant K d and the maximal binding A max ) were determined by nonlinear fitting A ¼ A max · [L]/(K d +[L])(relation1)whereA is the absorbance at 405 nm and [L] the ligand concentration, by using the CURVE FIT software developed by Kevin Raner Software, Mt Waverley, Victoria, Australia. Additional details on the different experimental conditions are given in the figure legends. Fluorescence measurements Fluorescence experiments were conducted with a LS 50 Perkin–Elmer luminescence spectrometer. Spectra for FITC labeled proteins were obtained in 0.1 M KCl 20 m M Tris/ HCl buffer pH 7.4, with the excitation wavelength set at 470 nm. Fluorescence changes were deduced from the area of the emission spectra of fluorescein isothiocyanate (FITC) between 505 and 525 nm. The parameters K d (apparent dissociation constant) and A max (maximum effect) were calculated by nonlinear fitting of the experimental data points as for ELISA (relation 1), or by using the following equation (relation 2), DF ¼ 1/2 A max · [E] )1 {([E] + [L] + K d ) ) (([E] + [L] + K d ) 2 ) 4[E] · [L]) 0.5 }where [E] is the concentration of the fluorescent protein. The maximum fluorescence change (A max ) at infinite substrate concentration expressed as percentage variation from initial fluorescence: F1 – F°/F° * 100 was calculated by the relation F 1 – F°/F° ¼ A max /F° where F° and F1 are fluorescence intensities for zero and infinite ligand concen- trations, respectively. Analytical methods Protein concentrations were determined by UV absorbency using a Varian MS 100 spectrophotometer. Gelsolin domain concentrations were determined spectrophotomet- rically using values of A 280 (1 cm )1 ) ¼ 15.5 l M for G4–6, and 79 l M for G2. These extinction coefficients were Ó FEBS 2003 Calcium activation of gelsolin (Eur. J. Biochem. 270) 2237 calculated by tryptophan, tyrosine, and cysteine content [33]. Electrophoresis was carried out on 15% (w/v) polyacrylamide slab gels (SDS/PAGE 15%) according to Laemmli [34] and stained with Coomassie blue or observed by UV for fluorescent gels before staining. The 14–97 kDa molecular weight marker kits were from Biorad. Calcium concentrations in the calcium-EGTA buffer system were experimentally measured by fluorometry using indo 1 as indicator [35]. Results Cleavage of G4–6 domain by trypsin A recent detailed study [19] reported that the exposure of various tryptic cleavage sites in gelsolin is actin, and calcium, dependent. In fact, several investigations using namely circular dichroism and light scattering [9,12,15,18] showed the occurrence of major conformational changes in the regulatory C-terminal half of gelsolin upon calcium binding. In an initial experiment, we tested the susceptibility of the C-terminal domain to proteolysis. As shown in Fig. 1A, tryptic digestion give rise to two fragments, one of 30 kDa and the other of 16 kDa. In order to identify these fragments, we have labeled the unique cysteine residue (Cys645) located in domain G6 with 1,5-I-AEDANS. As this residue is buried in the native molecule [15,22], the labeling was carried out after proteolytic digestion and SDS unfolding (see Methods). Figure 1A shows that only the 16 kDa band is fluorescent. This result demonstrates that the cleavage occurs in the loop between G5 and G6 domain and is in accord with the unpublished results reported previously [19]. As depicted in Fig. 1B, the tryptic cleavage was faster in EGTA than in calcium, suggesting that the orientation between the two domains is different and the junction more accessible in EGTA. Calcium induced change in the G4–6 and G2 domains Conformation changes induced by calcium binding were monitored by two approaches. First, intrinsic tryptophan fluorescence of G4–6 domain was measured in the presence of increasing calcium concentrations (between 1 n M and 1m M ). An increase in fluorescence intensity was observed at submicromolar calcium concentrations (Fig. 2). In a second experiment, conformational changes were detected from the extrinsic fluorescence measurements of FITC-labeled G4–6 domain. A biphasic relationship was observed (Fig. 2) producing two transitions in fluorescence intensity, one at  1.5 l M , and another around 0.1 l M . These transitions reflecting conformational changes correlate well with the two binding sites (IIG5 and IIG6, K d ¼ 2and0.2l M , respect- ively) detected and measured by equilibrium dialysis [7]. Intrinsic fluorescence of G2 domain was also measured as a function of calcium concentration (Fig. 2 insert). A simple reduction of fluorescence was observed with half maximum change occurring at a calcium concentration of  15 l M . Interaction of G2 segment with G4–6 domain In the crystallographic model of gelsolin in the EGTA form [22], G2 is tightly interacting with G6 segment (Fig. 3). Two regions in G2 domain appear included in this interface. The Fig. 2. Effect of calcium on the G4–6 tryptophan and FITC labeled G4–6 fluorescence emission. Aliquots of calcium (0.1 m M solution) were added successively to unlabeled G4–6 or labeled G4–6 in 0.1 M KCl, 20 m M Tris buffer pH 7.4 in the presence of 0.1 m M EGTA. Changes in fluorescence intensities corresponding to tryptophan emission (s) or FITC emission (d) are plotted vs. free calcium con- centration expressed as pCa ¼ log(1/[Ca 2+ ]). Calcium concentrations were determined experimentally (see Material and methods). Inset: effect of calcium on the G2 tryptophan fluorescence emission. Fluo- rescence changes were plotted vs. free calcium concentrations. Fig. 1. Effect of calcium on susceptibility of gelsolin G4–6 domain to tryptic digestion. (A) Identification of the two fragments (30 kDa and 16 kDa) produced by proteolysis in the presence of 1 m M EGTA followed by 1,5-I-AEDANS labeling as described in Material and methods. Molecular weight markers (lane T). G4–6 digest chemically modified by IEADANS and revealed with Coomassie blue (lane 1) or upon UV lamp (lane 2). (B) Digestion of G4–6 domain by trypsin (trypsin/G4–6 ratio: 1/25 w/w) for 10 min molecular weight markers (lane T). G4–6 domain (46 kDa) before proteolysis (lane 1). G4–6 digest in the presence of 0.1 m M calcium (lane 2) and in the presence of 0.1 m M EGTA (lane 3). Molecular weight marker are phosphory- lase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa). 2238 E. Lagarrigue et al. (Eur. J. Biochem. 270) Ó FEBS 2003 sequence 203–225, including one of the actin-binding sites, is in interaction with the C-terminal a-helix of G6 domain. The sequence 159–193, including the second actin interface, appears also in interaction with G6 domain. Therefore, we tested the interaction of G4–6 domain with G2 domain by two independent methods. In ELISA experiments, G4–6 fragment was immobilized on the plastic microtiter plate and the binding of biotinyl- ated G2 domain was revealed by using alkaline phosphatase labeled streptavidin. Figure 4 shows that binding occurs in the presence of EGTA as well in the presence of calcium although a better affinity is observed in the former case. These data were confirmed by studies in solution using fluorescence measurements. G2 or G4–6 domains were labeled by FITC and increasing concentrations of G4–6 or G2 were added, respectively. Figure 5A shows a decrease in the fluorescence intensity of FITC labeled G2 domain in the presence of EGTA. Analysis of these data shows that the fluorescence intensity decrease, extrapolated to infinite concentration of G4–6, is low (4%). Therefore, only a rough estimation of the apparent K d (about 0.5 l M )canbe obtained. This result shows that G4–6 fragment interaction induces a conformational change in G2 domain in the presence of EGTA. Calcium was without effect (Fig. 5A), but it is known that calcium did promote important changes [11,36], which would explain the present results. Conse- quently, further analyses, using FITC labeled G4–6, were carried out. Labeled G4–6 was incubated with increasing concentrations of G2 (between 0 and 7.6 l M )andthe changes in fluorescence were monitored. Saturation curves were observed in the presence of EGTA or calcium and apparent K d s of 0.8 and 3.5 l M , respectively, were deter- mined (Fig. 5B and Table 1). These values and those obtained above from ELISA show a better interaction of G2 in the presence of EGTA than calcium. The differences in the absolute values observed between the two methods are likely to be due to the heterogeneous phases used in ELISA. More interestingly, a significant difference in the maximum fluorescence enhancement determined in EGTA and calcium (about 8% and 30%, respectively) possibly reflects changes in domain conformation and in interfaces produced under these two conditions. In order to deduce a more precise relationship between G2 interaction and G4–6 calcium-induced conformation, the maximum fluorescence enhancement extrapolated for infinite G2 concentrations was determined at various calcium concentrations. As shown in Fig. 6, a change in the fluorescence from EGTA to calcium states was observed in the 0.1 l M range. This important result suggests that the transition would be linked to the high affinity calcium site in G4–6 [7], that we now know to be IIG6 [3] (see Discussion). In addition, apparent K d values were also estimated from the same fluorescence experiments performed in the presence of different calcium concentrations. As shown in Fig. 6 insert, an apparent K d transition occurs around 15 l M , a value which is observed for the binding of calcium to G2 domain. Footprint of G2 on gelsolin G4–6 domain Two sites within the G2 domain appear involved in the G2- G4–6 interface in the presence of EGTA (Fig. 3). The interaction of these two sites (residues 159–193 and 203– 225) was first tested by ELISA. The interaction of G4–6 with the plate-coated peptides was revealed using specific antibodies to G4–6. The results summarized in Table 1 Fig. 3. X-ray crystallographic structure of gelsolin in the absence of calcium (Burtnick et al. [22]) showing the interface between the G6 C-terminal domain and G2. The G6 domain is coloured green and its C-terminal alpha helix is coloured blue. The G2 domain is coloured yellow and its sequences in contact with G6, sequences 159–193 and 203–225 are coloured red and purple, respectively. Fig. 4. Binding of gelsolin domain G2 with G4–6 monitored by ELISA. Coated G4–6 was reacted with the biotinylated G2 domain in the presence of 1 m M EGTA (d) or in the presence of 1 m M CaCl 2 (s)at the concentrations indicated. Binding was monitored at 405 nm using alkaline phosphatase labeled streptavidin. Percentage binding was plotted vs. G2 concentrations. Ó FEBS 2003 Calcium activation of gelsolin (Eur. J. Biochem. 270) 2239 show that the two peptides interacted with G4–6 in the presence of EGTA. In contrast, only the 153–193 peptide interacted in the presence of calcium (Table 1). These results were confirmed in solution. For this aim, FITC labeled G4–6 was mixed with each peptide supplemented with EGTA or calcium. In the presence of EGTA, changes in the fluorescence intensity (Fig. 7A,B) were obtained with the two fragments. A maximum fluorescence quenching of 4% and fluorescence enhancement of 4% were calculated for 159–193 and 203–225 peptides, respectively. When calcium is present in the medium, binding of peptide 153–193 to labeled G4–6 induces an important decrease in fluorescence intensity (15%) (Fig. 7). The last result demonstrates that the binding of the latter peptide causes a somewhat different conformational change in G4–6 domain. In contrast no effect is observed for the 203–225 fragment, accordingly to ELISA experiments. Discussion Since its discovery as an actin-depolymerizing factor, gelsolin has now been implicated in a number of important pathways such as apoptosis, oncogenic transformation, signal trans- duction and amyloidosis (reviewed [2]). All of these pathways are likely to involve calcium activation, the process by which various binding sites become (especially actin) available for interaction. In this paper we confirm that calcium causes a large conformational change in the C-terminal half of gelsolin (G4–6) that can be monitored in a number of different techniques. We also show that G2 undergoes a conformational change upon binding calcium by a site that has a slightly higher affinity than was previously assumed. This study also provides some details on the interface between these two gelsolin domains (G2 with G4–6) whose dissociation is pivotal in the activation of gelsolin. Calcium-dependent conformational changes in G4–6 That the C-terminal half of gelsolin (G4–6) binds calcium, leading to the activation of the whole gelsolin molecule Fig. 5. Binding of gelsolin G2 domain with G4–6 monitored by fluor- escence measurements. (A) Interaction of FITC labeled G2 domain (0.2 l M ) with G4–6 was carried out in 0.1 M KCl 20 m M Tris buffer pH 7.4. Change in fluorescence emission spectra of FITC was recorded at various G4–6 concentrations (0–2.1 l M ) in the presence of 1 m M EGTA (d)or1m M CaCl 2 (s). (B) Binding of G2 domain with FITC labeled G4–6 (0.3 l M ) determined by fluorescence. The experiment was carried out in 0.1 M KCl, 20 m M Tris buffer pH 7.4 supplemented with 1 m M EGTA (d)or1m M CaCl 2 (s). Fig. 6. Effect of calcium on the fluorescence of the FITC labeled G4–6/ G2 complex. Maximum fluorescence enhancement (% initial fluores- cence) extrapolated to infinite G2 concentration is plotted vs. free calcium concentration (pCa). Inset, apparent K d s for G2. Interactions with FITC labeled G4–6 are plotted vs. pCa. Table 1. Binding of G2 and derived peptide to G4–6. EGTA K d (l M ) Calcium K d (l M ) Fluorescence ELISA Fluorescence ELISA G2 0.5–0.8 0.3 3 1 159–193 1 1 2–3 2 203–225 3 3 None None 2240 E. Lagarrigue et al. (Eur. J. Biochem. 270) Ó FEBS 2003 through large structural changes is well established [8,9]. We have shown that a calcium sensitive proteolysis between G5 and G6 occurs in agreement with the structural data [3,15] that shows a long random coil connecting G5 to G6. Previous data from biochemical studies on G4–6 have detect two calcium binding site one within G4-5 (K d ¼ 2 l M ), the other within G5–6 (K d ¼ 0.2 l M )[7]. New data (Koloppan et al. 2003) collected with actin-free G4–6 identify calcium bound to G5 and G6 only, a finding in agreement with Choe et al. [3] who hypothesized that a third site, IIG4, became coordinated solely by G4 residues only when actin was bound (were this to be the case then IIG4 is a site with properties between a site I and a site II type). Taken together it seems that two sites are bound by G4–6 in the absence of actin, one of these, IIG5 has a moderate affinity (K d ¼ 2 l M ) in agreement of this study and previous work [7], the other IIG6 has a higher affinity. The slight difference observed between the present study (K d ¼ 0.1 l M ) and the previous study [7] who measured a K d of 0.2 l M , may be attributable to conformational differences in the site in the context of G4–6 compared to G5–6 or a degree of cocooperativity between calcium site (see below). A calcium-dependent conformational change in G2 A number of studies have concluded that calcium induces conformational changes within G2 [11,36]. A calcium binding site has been detected in G1–3 [12], and others [3,11] have located this site within G2 itself. However, this previous [11] study estimated a low affinity (K d ¼32 l M ) whereas we have estimated a slightly higher affinity (K d ¼15 l M ). We have also found a calcium-dependent conformational change in G2 this may explain why the reactivity of the thiol groups within G2 are calcium-sensitive [37,38]. The G2 G4–6 interface As expected we have found that G2 binds to G4–6 with high affinity (K d ¼0.5–0.8 l M )inEGTA,weakerbinding was evident in the presence of 1 m M calcium (Fig. 5). In addition a change in the interface occurs during calcium binding to IIG6 site (Fig. 6). The interaction between gelsolin C-terminal helix (residues 744–755) and the helix of G2 is well known. We have confirmed this interaction biochemically and found a calcium dependent interaction between G4–6 and the peptide 203–225 derived from G2. The calcium sensitivity of this interaction is probably due to IIG6 as the coordinating residues that comprise the IIG2 site are not all present within the peptide. In addition to this expected interaction, we have also detected binding of G4–6 to a second G2-derived peptide 159–193. This interaction is only marginally calcium sensitive and of higher affinity (Table 1). The EGTA structure [22] reveals that this peptide makes salt bridge contacts (R168–D669 and R169–D670) and that hydrophobic interactions also occur such as those between V170-V657, with G6. In summary, G2 binds to G4–6 through two distinct interfaces. Binding site 1 involves G2 203–225 and G6 744–755 and binding site 2 involves G2 region around R168-R69 and G6 D669-D670. ‘Unlatching’ and dissociation of the G2 and G6 connection If the last 23 amino-acids are removed from gelsolin mutants, the requirement for calcium for actin-binding is lessened but not abolished [39], similarly it has been determined that adseverin which naturally lacks the C-terminal helix has a similar calcium requirement than the helix minus gelsolin mutant [21]. Together with recent observations on the structure of gelsolin [3,36], our new data on binding site 2 is compatible with the following Fig. 7. Effect of calcium on the interaction of two sequences involved in the G2–G4–6 interface monitored by fluorescence. Binding of FITC labeled G4–6 (0.3 l M ) with two synthetic peptides derived from G2 domain: (A) sequence 203–225 and (B), sequence 159–193. The experiments were performed in 0.1 M KCl, 20 m M Tris buffer pH 7.4 supplemented with 1 m M EGTA (d)or1m M CaCl 2 (s). Ó FEBS 2003 Calcium activation of gelsolin (Eur. J. Biochem. 270) 2241 explanation. It is likely that G2 remains held to G4–6 through binding site 2 in the absence of site 1. Occupancy of IIG2 has been proposed to disrupt site 1 [36], by disruption of hydrophobic and salt bridges between G2 helix a1 and the C-terminal helix of G6. It is proposed that binding of IIG6 disrupts the salt bridges between D669 and R168, and between D670 and R169. Cooper- ativity between IIG2 and IIG6 has been suggested to occur as a result of the breaking of site 1 and site 2, as breakage of either connection frees up ligands to coordinate calciums in either type II site [3]. Calcium binding to whole gelsolin has been found to be cooperative [40] it is possible that this is due to coordination of calcium sites as is proposed for IIG2 and IIG6, in addition to the general opening of the molecule. Cooperation of the sites would also explain why the dissociation of G2 from G4–6 occurs at 100 n M calcium (Fig. 6), whereas we have measured a K d value of 15 l M , and others estimate 32 l M [11] for calcium binding to IIG2. Occupancy of IIG6 may indirectly alter IIG2 so that it too becomes a high affinity site. We measure the IIG6 K d to be 100 n M (Fig. 2) in agreement with this model. Acknowledgements This research was supported by grants from AFM. References 1. Sun, H.Q., Yamamoto, M., Mejillano, M. & Yin, H.L. 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(1986) Characterization of the Ca 2+ -induced conformational change in gelsolin and identification of interaction regions between actin and gelsolin. Biochemistry 25, 3859–3867. 39. Kwiatkowski, D.J., Janmey, P.A. & Yin, H.L. (1989) Identifica- tion of critical functional and regulatory domains in gelsolin. J. Cell Biol. 108, 1717–1726. 40. Gremm, D. & Wegner, A. (1999) Co-operative binding of Ca 2+ ions to the regulatory binding sites of gelsolin. Eur. J. Biochem. 262, 330–334. Ó FEBS 2003 Calcium activation of gelsolin (Eur. J. Biochem. 270) 2243 . number of repeated domains; six in the case of gelsolin itself, adseverin and villin, and three in capG, fragmin and severin (for a review, see [2]). The binding of calcium to gelsolin and to actin bound. calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule. Keywords: actin; gelsolin; cytoskeleton; severing; calcium activation. Gelsolin is a. Co-operation of domain-binding and calcium-binding sites in the activation of gelsolin Emeline Lagarrigue 1 , Sutherland K. Maciver 2 , Abdellatif Fattoum 3 , Yves Benyamin 1 and Claude