Binding of gelsolin domain 2 to actin An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin Celine Renoult 1 , Laurence Blondin 1 , Abdellatif Fattoum 2 , Diane Ternent 3 , Sutherland K. Maciver 3 , Fabrice Raynaud 1 , Yves Benyamin 1 and Claude Roustan 1 1 UMR 5539 (CNRS) Laboratoire de Motilite ´ Cellulaire (Ecole Pratique des Hautes Etudes), Universite ´ de Montpellier, France; 2 Centre de Recherches de Biochimie Macromole ´ culaire, Montpellier, France; 3 Genes and Development Group, Department of Biomedical Sciences, University of Edinburgh, Scotland It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin’s severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule. Keywords: actin; actin-binding proteins; cofilin; gelsolin. The organization of the actin microfilaments in cells is dynamic and is quickly rearranged in response to extra- cellular signals. Gelsolin is one of the members of a family of proteins (e.g. severin, villin), that is essential for microfilament remodelling [1–3]. There are two forms of gelsolin which differ in their N-terminal extremities. One is specifically located in the blood and acts with vitamin D-binding protein to accelerate clearing of actin from the circulation [4], while the other form is intracellular. In vitro, gelsolin interacts with G- and F-actins, promotes nucleation and both severs and caps actin filaments. Cofilin belongs to another family of actin-binding proteins that also severs actin filaments and increases polymerization dynamics [5]. Despite a lack of sequence homology between the cofilin and gelsolin families the fold adopted by each of gelsolin’s 130 amino-acid subdomains [2] is similar to the actin depolymerizing factor (ADF)/cofilin family fold [6]. In contrast with cofilin, gelsolin does not appear to be essential for viability in the organisms where this has been tested, probably due to the expression of related genes such as adseverin/scinderin [7], but gelsolin is specifically required for rapid movement of various dynamic cells [8]. Thus, gelsolin over-expression in fibroblasts leads to enhanced cell motility [9,10]. Domains 1–3 (S1–3) are sufficient for capping and severing, while the C-terminal half of the molecule is directly implicated in calcium regulation. In particular, gelsolin domain 1 (S1) interacts both with monomeric actin, and with the barbed end of the actin filaments inhibiting polymerization. S2, in contrast, preferably binds to the side of the actin filament. Severing activity seems to require the binding of S2 to the filament, followed by interaction of S1 between two adjacent actins along the filament axis [11]. The tertiary structure of whole gelsolin in the inactive Ca 21 free state has been determined [2], as has S1 in complex with actin [11], gelsolin S4–6 [12], severin domain 2 [13,14] and villin domain 2 [15]. The structure of each gelsolin domain shows a surprising similarity to the cofilin fold [6]. Therefore it is possible to hypothesize that S2 binds actin in the same manner as cofilin [16]. The solution of the gelsolin structure [2], showed that when S1 is in position according to the G-actin–S1 model [11], S2 is not in contact with actin. This might suggest a reorientation of S1 : S2 interfaces so that S2 could contact both of the binding sites on the same actin unit in the filament to which S1 is joined [12]. In addition, by studying the S2–6 interaction with F-actin, McGough et al. [17] showed that the S2 –3 position on F-actin is similar to the actin-binding domain of a-actinin. Robinson et al. [12] presented a model for gelsolin interaction based on the Note: web pages are available at http://www.ephe.univ-montp2.fr, and http://www.bms.ed.ac.uk/research/smaciver/index.htm. Note: A gelsolin amino-acid numbering system based on the plasma human gelsolin [1], in which S1 is defined as extending from Pro39 to Tyr133 and S2 as being Gly137 to Leu247 [2], is used is this report. Correspondence to C. Roustan, UMR 5539(CNRS) UM2 CC107, Place E. Bataillon 34095 Montpellier Cedex 5, France. Fax: 133 04 67 14 49 27, E-mail: roustanc@crit.univ-montp2.fr (Received 14 June 2001, revised 28 September 2001, accepted 4 October 2001) Abbreviations: S1–6, the six repeated segments of gelsolin; ADF, Actin depolymerizing factor; 1,5-I-AEDANS, N,-iodoacetyl-N 0 -(sulfo- 1-naphthyl)-ethylenediamine; ELISA, enzyme-linked immunosorbant assay; FITC, fluorescein 5-isothiocyanate; G-actin, monomeric actin; F-actin, filamentous actin; EEDQ, N-ethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline. Eur. J. Biochem. 268, 6165–6175 (2001) q FEBS 2001 determination of the S4–6 actin structure. They suggested that changes in the structure of S1 –3 must occur to allow S2 to interact with the side of actin filament. Finally from mutagenesis and structural data, Puius et al. [14] proposed a model for S2 interaction in which 168RRV170 and 210RLK 212 are determinant in F-actin binding. In this report, we investigated the gelsolin S2 : actin interface. In particular, we focused on the comparison between respective locations of gelsolin and cofilin on actin filament and evidenced major differences in the interfaces. MATERIALS AND METHODS Proteins and peptides Rabbit skeletal muscle actin was isolated from acetone powder [18], and stored in buffer G (2 m M Tris, 0.1 mM CaCl 2 0.1 mM ATP pH 7.5). Actin was selectively cleaved by Staphylcoccus aureus V8 protease [19] and thrombin [20] and the fragments obtained were isolated by electroelution as described previously [19]. Human gelsolin domain 2 (S2) was produced in Escherichia coli, BL21(pLysS) carrying a vector containing a cDNA encod- ing residues including 151 – 266, the S2 repeat [21]. The bacteria were grown in 1-L flasks with 2 Â TY medium with ampicillin (150 mg : mL 21 ) and induced with isopropyl thio- b- D-galactoside (final concentration 1 mM) when the culture reached D 600 ¼ 0.5. Cultures were then grown for a further 4hat378C and the cells collected by centrifugation. The bacteria were lysed by repeated freeze–thaw cycles with sonication. Supernatant containing the S2 protein was applied to a DE52 column and purified further by hydroxyapatite chromatography. The concentration of S2 was determined spectrophotometrically assuming 1 A 280 ¼ 79 mM [21]. Antibodies directed towards gelsolin fragments 159–193 and 203–225 or actin sequences 75–105 and 285–375 were elicited in rabbits [22]. The antibodies directed to the actin sequences were selectively purified by affinity chromato- graphy [23]. Anti-IgG antibodies labelled with alkaline phosphatase were from Sigma. Synthetic peptides derived from actin and gelsolin sequences were prepared on a solid phase support using a 9050 Milligen PepSynthesizer (Millipore) according to the Fmoc/tBu system. The crude peptides were deprotected and purified thoroughly 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, UK), were in line with the expected structures. Peptides were labelled at the cysteine residue with N-iodoacetyl-N 0 -(sulfo-1-naphthyl)-ethylenediamine (1,5-I- AEDANS) or at amino groups by fluorescein 5-isothio- cyanate (FITC) [24,25]. Excess reagent was eliminated by sieving through a Biogel P2 column equilibrated with 0.05 M NH 4 HCO 3 buffer pH 8.0. Actin and gelsolin S2 domain were labelled by FITC as described elsewhere [25]. Excess reagent was eliminated by chromatography on a PD10 column (Pharmacia) in 0.1 M NaHCO 3 buffer pH 8.6. Actin was specifically labelled at cysteine 374 by 1,5-I- AEDANS [24]. Cross-linking experiments Actin (1 mg : mL 21 ) and gelsolin fragment 159–193 (0.1 mg : mL 21 ) were incubated with 2.5 mM N-ethoxy- carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; Sigma) in 100 m M Mops pH 7.0 at 22 8C. The cross-linking reaction was allowed to proceed for 45 min, and stopped by the addition of 100 m M 2-mercaptoethanol. The cross-linked species were separated and analysed by SDS/PAGE and immunoblotting. Immunological techniques The enzyme-linked immunosorbent assay (ELISA) [26], that was previously described in detail [27], was used to monitor interaction of ligands with coated peptides or actin. Actin (0.5 mg : mL 21 ) or peptides (5 mg : mL 21 )in50mM 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/50 mM Tris buffer pH 7.5. Experiments with coated peptides were performed in 0.15 M NaCl, 10 mM phosphate pH 7.5. Binding was monitored at 405 nm using alkaline phosphatase-labelled anti-IgG antibodies (1 : 1000) or alkaline phosphatase-labelled streptavidin (1 : 1000). Con- trol assays were carried out in wells saturated with gelatin and gelatin hydrolysate used alone. Each assay was con- ducted in triplicate and the mean value plotted after sub- traction 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 1 [L]) where A is the absorbance at 405 nm and [L] is the ligand concentration, by using the CURVE FIT software developed by K. Raner Software (Victoria, Australia). Additional details on the different experimental conditions are given in the figure legends. Western immunoblots were performed as described previously [28]. The immunoblots were revealed using alkaline phosphatase. Fourier transform IR measurements Fourier transform IR spectra were recorded using an IFS 28 Bruker spectrometer. Samples were placed in a horizontal ATR plate and the spectra recorded at room temperature. The peptide was analysed at a concentration of 5 mg : mL 21 in 10 mM phosphate buffer pH 7.5. A total of 500 scans were accumulated in the 1800–1500 cm 21 range. The Bruker OPUS/IR 2 program was used for spectrum analysis (second derivative). Fluorescence measurements Fluorescence experiments were conducted using a LS 50 Perkin-Elmer luminescence spectrometer. Spectra for 1,5-I- AEDANS or FITC were obtained in 50 m M Tris/HCl buffer pH 7.5, with the excitation wavelength set at 340 and 470 nm, respectively. Fluorescence changes were deduced from the area of the emission spectra of FITC between 480 and 500 nm. The parameters K d (apparent dissociation constant) and A max (maximum effect) were calculated by nonlinear fitting of the experimental data points. The number of binding sited (n ) and the affinity constant K a were also determined by another approach [29,30]. The 6166 C. Renoult et al. (Eur. J. Biochem. 268) q FEBS 2001 following relationship was then used: 1/ð1 2 XÞ¼K a ðC/ ðXEÞ 2 nÞð1Þ where C and E are total concentrations of peptide and actin, respectively, and X is the relative fluorescence change A/A max (corresponding to the fraction of peptide bound to actin). A plot of 1/(1 – X) ¼ vs. C/(XE) (Eqn 1) was drawn. The plot gives the number of binding sites which is the value of C/(XE) for 1/(1–X) ¼ 0. The slope of the same curve directly gives the value of the affinity constant. Analytical methods Protein concentrations were determined by UV absorbency using a Varian MS 100 spectrophotometer. Electrophoresis was carried out on 12.5% (w/v) polyacrylamide slab gels (SDS/PAGE 12.5%) according to Laemmli [31] and stained with Coomassie blue. The 14–97 kDa molecular weight marker kits were from Biorad. RESULTS Several investigations [32–35] have suggested the parti- cipation of gelsolin S2 sequences (and homologue S2 equivalents) within residues 197 – 226 (including the long helix of the domain) and within residues 161–172 (including the A strand and the AB loop in S2) in the interaction with F-actin. We have investigated subdomain 1 of actin, which possess accessible sequences in F-actin in order to delimit the interface of gelsolin S2 with the actin filament. In an initial experiment, we tested the ability of a sequence covering the helix of S2 to interact with actin. The conformation of the synthesized peptide (sequence 203–225) was checked in aqueous solution by Fourier transform IR. The IR spectrum of the peptide is charac- terized by the presence of a band at 1645 cm-1 (data not shown) suggesting an unordered conformation [36]. A similar result has already been observed for the corre- sponding peptide in cofilin [16]. Binding was tested by fluorescence measurements. In a first experiment, we have labelled actin at Cys374 with dansyl and the 203–225 peptide with FITC. The excitation was fixed at 340 nm and the fluorescence emission monitored between 460 and 480 nm. The fluorescence was corrected for the contribution of the FITC-labelled peptide alone. In this experiment, we observed a quenching of dansyl fluorescence emission (data not shown) that could be interpreted by energy transfer between the two chromophores and/or changes in the environment of Cys374 occurring during actin–peptide complex formation. In a second approach, FITC-labelled actin was incubated in the presence of increasing concentrations of 203 – 225 peptide (0–19 m M). The results shown in Fig. 1 indicate change in the FITC fluorescence induced by complex formation. The shape of the curve shows that the binding takes place in a saturable manner with an apparent K d of 5 mM. These experiments confirm the results of van Troys et al. [33] which implicate the sequence 197–226 in the gelsolin : actin interface. A second gelsolin S2 : actin interface is located in the N-terminal part of S2. In order to delimit the footprint of this gelsolin part on the actin structure, a peptide covering the 159–193 sequence was synthesized. Its conformation in an aqueous solution was studied by IR in the amide 1 region. The second derivative of the spectrum (Fig. 2), character- ized by a major band at 1629 : cm 21 associated with a band at 1680 : cm 21 suggests the presence of an antiparallel beta sheet structure [36]. In the corresponding region of gelsolin S2, crystallographic data reported the occurrence of three antiparallel strands [37]. The interaction of the 159–193 peptide with actin was documented by three independent assays. Actin was treated with EEDQ in the presence of peptide 159–193 and analysed by gel electrophoresis. A typical protein band pattern is shown in Fig. 3. The EEDQ treatment yields a new product with an apparent molecular weight of 47 kDa, which is not present with actin alone. This cross-linked product corresponds to the actin–peptide complex. Fig. 2. Structure of the synthetic gelsolin peptide 159 –193. Second derivative of the IR Fourier transform spectrum. Amide I region of the IR spectrum was observed in 10 m M phosphate, pH 7.5. Fig. 1. Binding of FITC-labelled G-actin with gelsolin 203 –225 peptide. Interaction of FITC-labelled G-actin at Lys61 (0.7 m M) with gelsolin 203–225 peptide was monitored by fluorescence. Changes in the intensity of the fluorescence emission spectra of FITC were recorded at various peptide concentrations in buffer G pH 7.5. Excitation was fixed at 470 nm and emission between 510 and 530 nm. q FEBS 2001 The actin gelsolin domain22 interface (Eur. J. Biochem. 268) 6167 The binding of the peptide to actin was also tested by ELISA. Its interaction with coated G-actin was revealed by using specific antibodies to sequence 159–193 within gelsolin S2. The results presented in Fig. 4A show that the peptide binds to G-actin with an apparent K d of < 2 mM. These data were confirmed in solution using fluorescence experiments. G-actin labelled with FITC was incubated in the presence of increasing 159– 193 peptide concentration and the changes in fluorescence were monitored. The saturation curve observed suggests a specific interaction with a K d of 2 mM. A stoichiometry of < 1 mole peptide per mole G-actin was also estimated (Fig. 4C). A similar experiment was performed with dansylated F-actin at Cys374 (Fig. 4B inset). The interaction induces a fluorescence quenching of the chromophore (K d ¼ 2 mM). Determination of the 159–193 peptide/actin interface Two approaches were used for identification of large fragments of actin to which gelsolin 159–193 peptide could be cross-linked by EEDQ. They involved the electrophoretic and immunological analysis of the cross-linked products formed either on proteolysis of the complex by V8 protease or on cross-linking of the 159–193 peptide to actin after digestion by thrombin. Digestion of actin by V8 protease gives two major fragments [19] of 31 and 16 kDa (1 – 225 and 226–375 sequence, respectively). As shown in Fig. 5, digestion of the cross-linked actin peptide complex reveals two faint bands at 33 kDa and 46 kDa which are missing from the controls. They can be stained by both anti-actin (directed towards sequence 75–105) and anti-gelsolin Fig. 4. Binding of gelsolin fragment 159 –193 with actin. (A) Interaction of gelsolin fragment monitored by ELISA. Coated G-actin was reacted with the gelsolin fragment at the concentrations indicated. Binding was monitored at 405 nm, using specific anti-gelsolin antibodies. (B) Interaction of FITC-labelled actin (0.7 m M) with gelsolin fragment was monitored by fluorescence. Changes in the intensity of the fluorescence emission spectra of FITC were recorded at various peptide concentrations (0–2.5 m M) in buffer G pH 7.5 supplemented with 50 m M KCl. Inset, Binding of gelsolin peptide to dansylated F-actin (1 m M) determined by fluorescence. The experiment was carried out in buffer F pH 7.5. (C) Quantitative analysis of the data in (B) for the interaction between G-actin and 159 –193 peptide was performedbyplotting1/(12 X) vs. C/(XE) where C is the concentration of peptide expressed in m M and E is the concentration of G-actin fixed at 0.7 m M. X, the binding ratio, was determined as described in Materials and methods. Fig. 3. Cross-linking pattern of actin –gelsolin peptide 159– 193 complex by EEDQ. The cross-linking reactions are performed as reported in Materials and methods. SDS/PAGE was carried out on a 12.5% acrylamide gel and then stained with Coomassie blue. Molecular mass markers (lane 1), actin alone (lane 2) and actin–peptide complex (lane 3) treated by EEDQ. 6168 C. Renoult et al. (Eur. J. Biochem. 268) q FEBS 2001 antibodies. These results show that the 159– 173 peptide is cross-linked to the 1–225 fragment of actin. The EEDQ-induced covalent complex between gelsolin peptide and thrombic digest of actin produces a 32-kDa band resulting from the covalent association between the 27 kDa fragment of actin (114–375 sequence) and the gelsolin fragment. This conclusion is supported by the fact that this band can be revealed by anti-gelsolin and anti-actin antibodies (directed towards 285–375 sequence) (Fig. 6) These results reveal that the cross-linking reactions impli- cate the residues within the 114–225 sequence of actin. Two large purified actin fragments [19,20] derived from thrombic and V8 protease digestion of actin (114–375 and 226 – 375 fragments) were tested for their possible interaction with 159–193 peptide by ELISA. The results shown in Fig. 7 indicate that both large fragments interacted with the gelsolin peptide. However binding to the 114–375 fragment was of higher affinity (apparent K d ¼ 1.8 mM) that binding to the 226–375 fragment (apparent K d ¼ 10 mM). Therefore, these results locate the actin site in central and C-terminal parts of actin. Identification of amino acid sequences implicated in the interfaces between actin and 159–193 fragment In the N-terminal extremity, the sequence 18–28 was previously show to be involved in gelsolin S2–3 domains binding [38]. We tested here the ability of the sequence to interact with the 159 –193 peptide. ELISA experiments in which 18–28 peptide was coated to plastic showed no Fig. 5. Analysis of the cross-linking between the gelsolin fragment 159–193 and actin with EEDQ after protease V8 digestion. The cross- linking reactions followed by a limited digestion by the V8 protease were carried out as described in Material and methods. Proteolysed material was analysed by 15% SDS/PAGE. Molecular mass markers (lane 1), actin treated by EEDQ (lane 2), gelsolin fragment 159–193-actin complex treated by EEDQ (lane 3). (A) SDS/PAGE stained by Coomassie blue. (B) Immunoblot revealed by specific antigelsolin antibodies. (C) Immunoblot revealed by specific anti-actin antibodies directed towards 75–105 sequence. Fig. 6. Analysis of the cross-linking between gelsolin fragment 159–193 and a thrombin digest of actin with EEDQ. After digestion of actin by thrombin, the cross-linking reaction with EEDQ was conducted as described in Material and methods. Proteolysed material was analysed by 17% SDS/PAGE. Molecular mass markers (lane 1), thrombic digest of actin (lane 2), thrombic digest of actin treated by EEDQ (lane 3), mixture of gelsolin fragment 159–193 and thrombic digest of actin treated by EEDQ (lane 4) and gelsolin fragment 159–193 treated by EEDQ (lane 5). (A) SDS/PAGE stained by Coomassie blue. (B) Immunoblot revealed by specific anti-gelsolin antibodies. (C) Immunoblot revealed by specific anti-actin antibodies directed towards 285–375 sequence. q FEBS 2001 The actin gelsolin domain22 interface (Eur. J. Biochem. 268) 6169 binding of the gelsolin fragment (Fig. 8A). The results indicate that this N-terminal part of actin may bind to another regions of S2. In addition, no interaction can be detected with the 85–103 sequence located near the 18–28 sequence at the surface of actin subdomain 1. A first interface was determined in a central region of actin as evidenced by cross-linking experiments (sequence 114–225). Two peptides belonging both to the 114–225 sequence and exposed regions of subdomain 1 were tested (112 – 125 and 119–132 peptides). Interaction of the 159–193 fragment with the coated peptides was revealed using specific anti-gelsolin antibodies. The results reported in Fig. 8A indicate that only peptide 119–132 interacted with a K d of 2.9 mM. The binding of the 119 –132 peptide was confirmed in solution. To perform such an experiment gelsolin 159–193 fragment was labelled with FITC and mixed with 112–125 and 119–132 actin peptides. As shown in Fig. 8B, the 119–132 peptide does not perturb FITC. In contrast the 112–125 peptide induces a fluorescence decrease of the label, but the corresponding binding is very weak (K d . 50 mM). Therefore, to test the 119–132 sequence, corresponding peptide was synthesized with an extra cysteine at the N-terminal extremity, then labelled with 1,5-I-AEDANS. The binding of the gelsolin fragment increases the dansyl fluorescence (Fig. 8C). Analysis of the saturation curve shows binding parameters which confirm the ELISA results (K d ¼ 2 mM). A second interface was then evidenced in the C-terminal part of actin. The more accessible sequences in this region were first investigated by ELISA. One corresponds to the helix 338–348, and the other to two helices and one turn located in the 356–375 sequence. The corresponding peptides (339–349, 347–365, 356–375 and 360–372) were coated to plastic. We observed (Fig. 8A and Table 1) that only peptides 356–375 and 347–365 interacted signi- ficantly with the gelsolin fragment. The activities of overlapping peptides within the C-terminal of actin towards Fig. 7. Interaction of gelsolin peptide 159–193 with two large C-terminal fragments of actin. Actin (0.5 mg : mL 21 )(W) or two actin fragments (0.5 mg : mL 21 ) derived by protease v8 digestion (B) (actin sequence 226 –375) or thrombic digestion (X) (actin sequence 114–375) were coated to plastic. Increasing concentrations of gelsolin fragment 159–193 were added in buffer containing 0.15 M NaCl, 1% BSA 10 m M phosphate pH 7.5, 0.1 mM dithiothreitol. Binding was detected by using anti-gelsolin antibodies and was monitored at 405 nm. Fig. 8. Determination of actin sequences involved in the gelsolin fragment 159–193 : actin interface. (A) Interaction of the gelsolin fragment with various actin synthetic peptides monitored by ELISA. Actin peptides [sequences 18–28 (O), 112 –125 (A), 119 –132 (W), 339–349 (B) and 356–375 (X)] were coated to plastic at a concentration of 5 mg : mL 21 . ELISA was carried out as in Fig. 7. (B) Interaction of FITC-labelled gelsolin fragment 159–193 with actin synthetic peptides of sequences 84–103 (O), 112–125 (W), 347–365 (A), 360–372 (B) and 356–375 (X). Experiments were carried out with FITC-labelled peptide (2 m M)in50mM Tris buffer pH 7.6. (C) Interaction of dansylated synthetic peptides derived from actin sequence to gelsolin fragment 159 –193. In creasing concentrations of gelsolin fragment were added to peptide 119–132 (X), 347–365 (W) and 360–372 (B) in 0.05 M Tris buffer pH 7.6. 6170 C. Renoult et al. (Eur. J. Biochem. 268) q FEBS 2001 FITC-labelled gelsolin fragment were finally tested by fluorescence. As shown in Fig. 8B, only 356 –375 peptide interaction can be characterized by this method. Finally, dansylated peptides 347–365 and 360–372 were tested. The peptide interaction of 348–365 peptide with the gelsolin fragment was evidenced (Fig. 8C). All of these facts suggested corresponding interfaces to be located in the C-terminal part of actin. Competition between the N-terminal part of gelsolin S2 and cofilin Van Troys and colleagues [16] have proposed that cofilin and gelsolin S2 share a similar target site on the filament. To show the overlapping of these two proteins on the actin surface, competition between cofilin and the gelsolin 159–193 fragment was studied by ELISA. G-actin was coated to plastic and increasing concentrations of gelsolin peptide were added to a fixed concentration of cofilin (0.8 m M). The binding of the ligand used at a fixed con- centration was monitored using the corresponding cofilin- specific antibodies. The results presented in Fig. 9 indicate that a ternary complex actin–cofilin–gelsolin peptide might occur as the binding of cofilin decreases only partially to < 45% as the gelsolin peptide concentration is increased. Footprint of gelsolin S2 on actin To confirm the ability of sequences 119–132, 18 –28 and 356–375 to bind gelsolin, experiments were performed with the entire domain 2. First, although this domain appears to bind preferentially to F-actin, we tested the possible interaction with G-actin. For this purpose, gelsolin domain 2 was labelled with FITC and increasing concentrations of Fig. 10. Binding of gelsolin domain S2 to actin. (A) FITC labelled gelsolin domain S2 (0.26 m M) was mixed with 0 –4 mM G-actin in G-buffer pH 7.5. Changes in the emission spectra were reported vs. actin concentrations. (B) Increasing concentrations (0–7 m M)of gelsolin domain S2 were incubated with several fluorescent peptides derived from actin sequence [dansylated peptide 18–28 (B), dansylated peptide 119–132 (X) and FITC-labelled peptide 356–375 (W)]. Fluorescence changes were reported vs. S2 concentrations. (C) Effect of short actin peptide on the interaction of gelsolin S2 to G-actin. FITC- labelled gelsolin S2 (0.26 m M) was mixed with increasing concentration of G-actin (final concentration, 2.5 m M) in G-buffer pH 7.5 and the spectrum was recorded between 510 and 530 nm. Then, increasing concentrations of actin peptides [peptide 1–10 (A), 18 –28 (B), 119–132 (W), 339–349 (O) and 356–375 (X)] were added and corresponding spectra were recorded. The binding is expressed as binding relative to that without peptides. Fig. 9. Competition binding study between gelsolin fragment 159–193 and cofilin. The binding of cofilin (0.8 m M) to coated G-actin in 150 m M NaCl, 10 mM phosphate buffer pH 7.5 supple- mented with 1% BSA and 0.1 m M dithiothreitol was performed in the presence of increasing gelsolin fragment concentrations (0–24 m M). Binding was detected by using anti-cofilin antibodies and was monitored at 405 nm. q FEBS 2001 The actin gelsolin domain22 interface (Eur. J. Biochem. 268) 6171 actin were added. As shown in Fig. 10A, we observed changes in the fluorescence intensity. Analysis of these data give an apparent K d of < 5 mM. The interactions evidenced for gelsolin domain 2 with the three actin peptides (peptides 18–28, 119 –132 and 356–375 [39]) labelled, either with dansyl or FITC (Fig. 10B), are in agreement with the above results. Finally competitions for the binding of S2 and actin peptides to G-actin were also performed (Fig. 10C). We observed the dissociation of actin–S2 complex by peptides 119–132 and 356–375. However peptides 18–28 and 338–348 had no effect. DISCUSSION The actin-binding site on S2 S2 (137–247) contains gelsolin’s initial F-actin binding site prior to severing/capping microfilaments [40], but the orientation of contacting residues and to a lesser extent the identity of these residues within S2 is less certain. The first 10 residues of S2 in addition to S1 is the minimal requirement for filament severing [41]. The standard explanation for this is that a very weak F-actin binding region exists within these 10 residues; however, additional F-actin affinity afforded by other residues of S2 is necessary for full severing [42]. A peptide derived from villin equi- valent to residues 159–171 of human gelsolin S2 was found to bind F-actin and to bundle it if an extra cysteine residue was placed at the C terminus of the peptide allowing dimerization by disulfide cross-linkage [35]. A similar peptide (159–174) from gelsolin itself has also been shown to bind F-actin [34], with a K d of 4 mM. Residues 198–227 of human gelsolin bind actin, cross-link to F-actin and compete with S2–3 for binding to F-actin [33]. A deletion study [32] concluded that a mutant 173–266 was not able to bind actin. Additional data on the actin-binding site on S2 comes from mutational studies in which the importance of two sites (168–171 and 210–213) were highlighted [14]. It is possible that the introduction of such mutations may alter binding by subtle disruption of the structure and so a more convincing approach has been taken by Southwick [43] in which the non-actin binding S2 equivalent of the gelsolin- related protein CapG was transformed into an actin binding region by the substitution of gelsolin 108 Leu2114 Gly, in the equivalent position of CapG thus indicating their likely importance in actin-binding. We too have confirmed the importance of the NH 2 -terminal portion of S2 in actin binding and report that gelsolin 203–225 peptide binds actin, as does 159–193. The comparatively weak binding and short length of peptide 203–225 has made the deter- mination of its binding site on actin and the stochiometry of Fig. 11. A model for the interaction of gelsolin with the actin filament. Our data suggest that S1 and S2 bind to the same actin monomer exposed at the barbed end of the filament after severing. S3 acts as a spacer connecting S2 to S4 which binds either to the diagonally opposed actin monomer ‘a’ or monomer ‘b’. We prefer monomer ‘a’ as this affords the shortest distance across the filament. S4 binds the actin monomer with a similar interface as S1. S5 and S6 do not, as far as is known, bind actin and may stick out from the filament as illustrated. Table 1. Summary of binding of gelsolin peptide 159–193 and cofilin to various parts of actin and whole actin in the F- and G-form by similar methods. Note that no K d value is given for cofilin binding to F-actin as the co-operativity of the interactions precludes this. ND, Not determined. Tested sequences Peptide 159–193 K d ELISA Peptide 159–193 K d fluorescence Cofilin K d Reference for cofilin Actin G 1.3 m M 2.0 mM 1.5 mM [51] Actin F ND 2.0 m M ?– 1–10 No binding ND No binding [51] 18–28 No binding ND 3 m M a 84–103 No binding No binding 1–2 mM a 112–125 No binding 50 mM 4 mM [51] 119–132 2.9 m M 2.0 mM 12 mM [51] 347–365 2 m M Binding 1 15 mM a 338–348 40 mM ND 4 mM a 360–372 . 30 mM No binding 2 mM a 355–375 2 mM 4 mM 2 mM a a L. Blondin, C. Renoult, Y. Bemyamin & C. Roustan, unpublished data. 6172 C. Renoult et al. (Eur. J. Biochem. 268) q FEBS 2001 interaction uncertain; however, we have no reason to believe that this is not simply 1 : 1. Furthermore we have located the site of interaction on the actin molecule. The S2-binding site on actin Van Troy and colleagues [33] have used sequence-specific actin antibodies to localize the site cross-linked to S2 peptide 198–227 and found that they could exclude residues 12–44, 228–257 and 354–375 from being the site of peptide binding. Our adjacent peptide S2 159–193 did not bind the C terminus of actin (360–372) either but we did measure a reasonable binding (K d 3–5 mM) to actin peptide 355–375 and to 347–365 (K d 2 mM) (Table 1). It is possible that both S2 and the antibody used by this group [33] are able to bind 355–375 of actin simultaneously. We measured tight binding (K d 1.8 mM) to 114–375 and weaker binding (K d 10 mM) to 226–375 of actin. As the affinity for S2 to the entire actin molecule is within this range (K d 1.4–7.9 mM) [14,32] perhaps there is no other region on the surface of the actin molecule that binds actin. This is not compatible with Puius et al. [14] who postulated a second monomer interface with the DNase1 binding loop of actin in subdomain 2. Pope et al. [44] have shown that DNase1 does not interfere with the binding of S2–3, but perhaps binding can occur through the first actin binding site in S2 [14]. The S2 actin interface compared with ADF/cofilin The similarity in structural fold between all gelsolin domains and between these and the ADF/cofilin fold has enticed some to compare the latter to both S1 [6,45] and S2 [16], despite the fact that important differences exist in the manner in which S1 and S2 bind actin and that they bind different, nonoverlapping sites [44]. S1 binds G-actin primarily, S2 binds F-actin exclusively (in salts) and the ADF/cofilins bind both G- and F-actin but in a significantly different manner to either gelsolin family domain. The main feature of ADF/cofilin is the extreme co-operativity in F-actin binding [46] (a Hill coefficient of 3 has been measured). This is in marked contrast with the situation with S2 where no evidence for co-operation in F-actin binding was observed by many other studies [47]. Binding of S2, S2–3 found by Scatchard analysis to bind F-actin with a K d value of 1.44 mM [47]. S2 competes with a-actinin for actin binding [21,48]; however, only slight competition is evident between cofilin and a-actinin [49]. McGough and colleagues [50] suggest that a-actinin binds between two longitudinally associated actin monomers in the filament in line with the model proposed by Puius et al. [14] who suggest that S2 binds actin via two faces, one S1-like, the other encompassing 38–62 includes the DNase1 binding loop and 92–95. In this respect S2 is like ADF/cofilin as we have postulated a similar two-site scheme [51]. The fact that the gelsolin fold is so similar to the ADF fold with only tentative suggestions of homology [52,53] is all the more remarkable because despite the structural similarity and the fact that both gelsolin and ADF/cofilin are actin-binding proteins, the fold seems to form at least three quite distinct actin binding interfaces. Ultimately, structural solutions of both S2-decorated and ADF/cofilin-decorated F-actin will be required to establish the exact F-actin binding characteristics of these different protein families and how similar or otherwise they truly are. The orientation of S2 with respect to actin, and implications for gelsolin on the microfilamen t How the six gelsolin domains arrange themselves around the actin filament to sever and cap it remain controversial. We have characterized an S2-binding site on subdomain 1 of actin adjacent to but not overlapping that of the S1 site between subdomains 1 and 3 [11]. S1 plus a short peptide (Phe134–Gln160) running into S2 is sufficient for severing [41]. As this is likely to be brought about by weak F-actin binding by the peptide, and this region is so close to S1 it is probable that the N terminus of S2 binds subdomain1 of actin. We now report that 159–193 of S2 binds to regions within 119 –132 and 347 –375 of actin both towards the outer surface of the filament on subdomain 1. The actin monomer is generally flat, and in the standard orientation the actin monomer has it flat face presented. We have determined that S2 binds subdomain 1 on the lower edge and even perhaps ‘behind’ this flat face surface. This placement would explain the capping activity observed in S2 [32] as binding in this region would prevent monomer addition at the barbed end by blocking the longitudinal binding site between subdomain 1 and the DNase1 site of the incoming monomer. However our model is not easily reconcilable with that proposed by Puius et al. [39] who predict that S2 binds actin with a similar interface as S1 in addition to binding around the DNAse1 binding site in subdomain 2 of actin. The Puius model is attractive in that proposing two actin- binding sites explains why S2 causes oligomerization of actin [39], why S1 –3 binds two actin monomers [54], and fits a reconstruction of the S2–6 decorated filament. S2 produces oligomerization of actin monomers [39], and the peptide 159 –174 from S2 increases the rate of spontaneous actin polymerization [34] but does not increase elongation or the extent of final polymerization. One possible inter- pretation of these facts is that S2 binds two longitudinally associated monomers; however, it is also possible that S2 binding induces a change in the conformation of actin [55] to that of the F-monomer accounting for the tendency for oligomerization and polymerization. Our model (Fig. 11) is similar to that proposed previously by Pope et al. [44] in that we also propose that S3 connects S2 to S4 the ‘long-way around’ the microfilament (so that S4 binds the diagonally opposite actin monomer) and that both models place S1 and S2 on the same actin monomer. Where the present model differs is that we place S2 beside S1 on subdomain 1 of actin instead of on subdomain 2 and this requires S3 or parts of it to be more extended than the other domains. In the crystallographic solution of gelsolin, it is clear that S2 is connected to S3 by a relatively long linker region which in the absence of Ca 21 connects S2 to S3 by wrapping around S1. The position of S2 at the edge of subdomain 1 shortens the distance that S3 has to straddle S2 and S4. Major rearrangements between the domains must occur between the Ca 21 -free and Ca 21 -bound gelsolin [2,12]. There is presently little data to distinguish if S4, which binds actin [56] in a manner to S1 [12], binds the actin monomer (a) as shown (Fig. 11) or the monomer that would have been q FEBS 2001 The actin gelsolin domain22 interface (Eur. J. Biochem. 268) 6173 placed immediately under it (b); however, we prefer the model as shown as it seems that this would be the shortest route given how the backbone is positioned at the C terminus of S2. The positions of S5 and S6 relative to the capped filament are not known with any precision but are shown ‘sticking out’ from the filament as electron microscopic data from gelsolin S2–6-decorated microfilaments [17] indicate that this is possible. ACKNOWLEDGEMENT We thank P. McLaughlin for many valuable comments on the work. REFERENCES 1. Kwiatkowski, D.J., Stossel, T.P., Orkin, S.H., Mole, J.E., Colten, H.R. & Yin, H.L. (1986) Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. 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A.G (19 89) Expression of human plasma gelsolin in E coli and dissection of actin binding sites by segmental deletion mutagenesis J Cell Biol 10 9, 593–605 Khaitlina, S & Hinssen, H (19 97) Conformational changes in actin induced by its interaction with gelsolin Biophys J 73, 929 –937 Pope, B., Maciver, S & Weeds, A.G (19 95) Localization of the calcium-sensitive actin monomer binding site in gelsolin to. .. interaction with gelsolin Biophys J 73, 929 –937 Pope, B., Maciver, S & Weeds, A.G (19 95) Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium -binding sites Biochemistry 34, 15 83 15 88 . binding ND 3 m M a 84 10 3 No binding No binding 1 2 mM a 1 12 12 5 No binding 50 mM 4 mM [ 51] 11 9 1 32 2.9 m M 2. 0 mM 12 mM [ 51] 347–365 2 m M Binding 1 15. (sequence 11 4 22 5). Two peptides belonging both to the 11 4 22 5 sequence and exposed regions of subdomain 1 were tested (1 12 – 12 5 and 11 9 1 32 peptides).