Analogous F-actin Binding by Cofilin and Gelsolin Segment 2 Substantiates Their Structural Relationship
1997; Elsevier BV; Volume: 272; Issue: 52 Linguagem: Inglês
10.1074/jbc.272.52.32750
ISSN1083-351X
AutoresMarleen Van Troys, Daisy Dewitte, Jean‐Luc Verschelde, Mark Goethals, Joël Vandekerckhove, Christophe Ampè,
Tópico(s)Biotin and Related Studies
ResumoCofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047–1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366–369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369–373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin α-helix 112–128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2–3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics. Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047–1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366–369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369–373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin α-helix 112–128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2–3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics. Cofilin belongs to a family of actin modulating proteins that is widely distributed throughout eukaryotes. Homologs have been identified in vertebrates, plants, yeast, Drosophila,Caenorhabditis, Acanthamoeba, and Dictyostelium (Ref. 1Moon A. Drubin G.D. Mol. Biol. Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (226) Google Scholar and references therein). In vivo they are found in the highly motile periphery of the cell (2Bamburg J.R. Bray D. J. Cell. Biol. 1987; 105: 2817-2825Crossref PubMed Scopus (155) Google Scholar,3Aizawa H. Sutoh K. Tsubuki S. Kawashima S. Ishii A. Yahara I. J. Biol. Chem. 1995; 270: 10923-10932Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and overexpressing cofilin in Dictyostelium cells stimulates their motility (4Aizawa H. Sutoh K. Yahara I. J. Cell Biol. 1996; 132: 335-344Crossref PubMed Scopus (129) Google Scholar). Cofilin appears essential for yeast growth (5Iida K. Moriyama K. Matsumoto S. Kawasaki H. Nishida E. Yahara I. Gene (Amst.). 1993; 124: 115-120Crossref PubMed Scopus (123) Google Scholar, 6Moon A.L. Janmey P.A. Louie K.A. Drubin D.G. J. Cell Biol. 1993; 120: 421-435Crossref PubMed Scopus (201) Google Scholar) and is important during cytokinesis in Drosophila (7Gunsalus K.C. Bonaccorsi S. Williams E. Verni F. Gatti M. Goldberg M.L. J. Cell Biol. 1995; 131: 1243-1259Crossref PubMed Scopus (251) Google Scholar) and Xenopus (8Abe H. Obinata T. Minamide L. Bamburg J.R. J. Cell Biol. 1996; 132: 871-885Crossref PubMed Scopus (164) Google Scholar). Cofilin family members are multifunctional in vitro, displaying binding to both monomeric and filamentous actin. Cofilin and actin depolymerizing factor (ADF 1The abbreviations used are: ADF, actin depolymerizing factor; EDC, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide; P3, cofilin actin binding peptide (residues 102–130); PS1, gelsolin segment 1 actin binding peptide (residues 88–117); PS2, gelsolin segment 2 actin binding peptide (residues 198–227); S1, gelsolin segment 1; S2, gelsolin segment 2; S2–3, gelsolin segment 2 to 3; TFE, 3,3,3-trifluoroethanol; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid. 1The abbreviations used are: ADF, actin depolymerizing factor; EDC, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide; P3, cofilin actin binding peptide (residues 102–130); PS1, gelsolin segment 1 actin binding peptide (residues 88–117); PS2, gelsolin segment 2 actin binding peptide (residues 198–227); S1, gelsolin segment 1; S2, gelsolin segment 2; S2–3, gelsolin segment 2 to 3; TFE, 3,3,3-trifluoroethanol; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid. or destrin) are functionally equivalent, and the mechanism by which they enhance filament turnover has recently been addressed (9Carlier M.-F. Santolini J. Laurent V. Didry D. Yan H. Chua N.-H. Pantaloni D. Mol. Biol. Cell. 1996; 7: 546aGoogle Scholar, 10Weeds A.G. Pope B. Whytock S. Maciver S. Mol. Biol. Cell. 1996; 7: 202aGoogle Scholar, 11Carlier M.-F. Laurent V. Santolini J. Melki R. Didry D. Xia G.-X. Hong Y. Chua N.-H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1323Crossref PubMed Scopus (817) Google Scholar). In an important study, Carlier et al. (9Carlier M.-F. Santolini J. Laurent V. Didry D. Yan H. Chua N.-H. Pantaloni D. Mol. Biol. Cell. 1996; 7: 546aGoogle Scholar) provide evidence that members of the cofilin family act by accelerating the rate of treadmilling because they increase the rate of dissociation from the pointed end of the filament and consequently, by providing a large monomer pool, also the rate of association to the barbed end. Consistent with this notion is the observation that the rate of propulsion and/or the length of the tails of locomoting Listeria (i.e. the rate of turnover of filaments in the tail) is strongly dependent upon ADF (9Carlier M.-F. Santolini J. Laurent V. Didry D. Yan H. Chua N.-H. Pantaloni D. Mol. Biol. Cell. 1996; 7: 546aGoogle Scholar,12Rosenblatt J. Agnew B.J. Bamburg J.R. Mitchison T.J. Mol. Biol. Cell. 1996; 7: 545aGoogle Scholar, 13Rosenblatt J. Agnew B.J. Abe H. Bamburg J.R. Mitchison T.J. J. Cell Biol. 1997; 136: 1323-1332Crossref PubMed Scopus (190) Google Scholar). Carlier et al. (9Carlier M.-F. Santolini J. Laurent V. Didry D. Yan H. Chua N.-H. Pantaloni D. Mol. Biol. Cell. 1996; 7: 546aGoogle Scholar) show that in vitrothis activity results in a new steady state with a larger monomer pool (of G-actin and G-actin-ADF) that varies with pH. This explains earlier observations that show that these proteins mainly bind to the actin filament in a 1:1 molar ratio to actin protomers at near neutral pH, but at slightly more alkaline pH these proteins induce actin depolymerization to a larger extent (14Yonezawa N. Nishida E. Sakai H. J. Biol. Chem. 1985; 260: 14410-14412Abstract Full Text PDF PubMed Google Scholar, 15Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar). The interaction of cofilin and ADF with actin is inhibited by phospholipids and regulated by phosphorylation, making them prime candidates as stimulus responsive mediators of actin dynamics (16Yonezawa N. Nishida E. Iida K. Yahara I. Sakai H. J. Biol. Chem. 1990; 265: 8382-8386Abstract Full Text PDF PubMed Google Scholar, 17Davidson M.M.L. Haslam R.J. Biochem. J. 1994; 301: 41-47Crossref PubMed Scopus (71) Google Scholar). Phosphorylated ADF and cofilin are unable to bind to actin, and in vivo dephosphorylation of these proteins is correlated with cytoskeletal reorganization (18Agnew B.J. Minamide L.S. Bamburg J.R. J. Biol. Chem. 1995; 270: 17582-17587Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 19Moriyama K. Iida K. Yahara I. Genes to Cells. 1996; 1: 73-86Crossref PubMed Scopus (310) Google Scholar). A region in the COOH-terminal half of cofilin has been demonstrated to participate in actin binding. By peptide mapping of a cross-linked actin-cofilin complex and mutational analysis of cofilin, lysine residues 112 and 114 were shown to interact with actin (20Moriyama K. Yonezawa N. Sakai H. Yahara I. Nishida E. J. Biol. Chem. 1992; 267: 7240-7244Abstract Full Text PDF PubMed Google Scholar). These residues are part of the motif 111LKSKM115 that shows homology with actin binding motifs in other actin modulating proteins (21Vandekerckhove J. Vancompernolle K. Curr. Opin. Cell Biol. 1992; 4: 36-42Crossref PubMed Scopus (46) Google Scholar). A synthetic peptide patterned around these residues (Trp104-Met115) inhibits actin polymerization and induces depolymerization (22Yonezawa N. Nishida E. Iida K. Kumagai H. Yahara I. Sakai H. J. Biol. Chem. 1991; 266: 10485-10489Abstract Full Text PDF PubMed Google Scholar). However, the sequence formed by cofilin residues Asp122-Leu129 (homologous to the NH2 terminus of tropomyosin) has also been implicated in actin binding as the corresponding synthetic heptapeptide competes with cofilin for binding to actin (23Yonezawa N. Nishida E. Ohba M. Seki M. Kumagai H. Sakai H. Eur. J. Biochem. 1989; 183: 235-238Crossref PubMed Scopus (44) Google Scholar). Both amino acid sequences Trp104-Met115 and Asp122-Leu128 are highly conserved throughout the cofilin/ADF family. We hypothesized that these two neighboring motifs, which in previous studies were considered as separate actin binding entities (22Yonezawa N. Nishida E. Iida K. Kumagai H. Yahara I. Sakai H. J. Biol. Chem. 1991; 266: 10485-10489Abstract Full Text PDF PubMed Google Scholar, 23Yonezawa N. Nishida E. Ohba M. Seki M. Kumagai H. Sakai H. Eur. J. Biochem. 1989; 183: 235-238Crossref PubMed Scopus (44) Google Scholar), essentially are part of the same site. Therefore, we studied the properties of a chemically synthesized peptide that spans both sites of interest, and we show that both motifs cooperate and actually form one actin binding unit. These data are supported by the recently determined, three-dimensional structures of yeast cofilin and the cofilin homologs destrin and actophorin which all have a fold similar to segments of proteins of the gelsolin family (24Hatanaka H. Ogura K. Moriyama K. Ichikawa S. Yahara I. Inagaka F. Cell. 1996; 85: 1047-1055Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 25Fedorov A.A. Lappalainen P. Fedorov E.V. Drubin D.G. Almo S.C. Nat. Struct. Biol. 1997; 4: 366-369Crossref PubMed Scopus (94) Google Scholar, 26Leonard S.A. Gittis A.G. Petrella E.C. Pollard T.D. Lattman E.E. Nat. Struct. Biol. 1997; 4: 369-373Crossref PubMed Scopus (61) Google Scholar). Based on the, albeit weak, sequence similarity between cofilin and gelsolin segment 2 (S2), we performed experiments to show that both the actin binding peptide of cofilin and the intact protein compete for binding to F-actin with the filament binding domain of gelsolin (segment 2–3). Our competition data strongly suggest that the cofilin family and gelsolin S2 use a common structural topology to bind the actin filament and interact with a similar target site. At least in their F-actin binding properties, proteins of the cofilin family may thus be considered as functional homologs of segment 2 of gelsolin that have evolved to be differently regulated. We prepared rabbit skeletal muscle actin following the procedure of Spudich and Watt (27Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) and isolated it as Ca2+-ATP-G-actin by chromatography over Sephadex G-200 in G buffer (5 mm Tris-HCl, pH 7.7, 0.1 mmCaCl2, 0.2 mm ATP, 0.2 mmdithiothreitol, 0.01% NaN3). Actin was labeled with N-pyrenyliodoacetamide as described (28Kouyama T. Michashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (717) Google Scholar). We prepared human plasma gelsolin, digested it with α-chymotrypsin, and purified the obtained gelsolin fragments following the procedure of Bryan (29Bryan J. J. Cell Biol. 1988; 106: 1553-1562Crossref PubMed Scopus (112) Google Scholar). Cofilin was purified from porcine brain based on the protocol of Yonezawa et al. (30Yonezawa N. Nishida E. Maekawa S. Sakai H. Biochem. J. 1988; 251: 121-127Crossref PubMed Scopus (38) Google Scholar) with slight modifications. We used butyl-Sepharose as hydrophobic interaction column, followed by a hydroxyapatite column. This yielded a cofilin preparation only contaminated with a 25-kDa protein (M app) which we identified as phosphatidylethanolamine-binding protein. Attempts to purify cofilin further systematically resulted in the loss of the protein. We used the mixture whereby phosphatidylethanolamine-binding protein serves as an internal control since it does not interact with G- or F-actin. Cofilin peptides and the gelsolin segment 2 and segment 1 peptides were chemically synthesized on a model 431A peptide synthesizer (Applied Biosystems Inc., Foster City, CA), purified, checked for correct mass, and their concentration determined as in Ref. 31Van Troys M. Dewitte D. Goethals M. Carlier M.-F. Vandekerckhove J. Ampe C. EMBO J. 1996; 15: 201-210Crossref PubMed Scopus (120) Google Scholar. We synthesized the gelsolin peptide PS2 also with an extra cysteine at the NH2terminus and in this way were able to label it with 14C (using [14C]iodoacetamide). The cofilin peptide P3 was labeled with 14C on methionine residue 115 using [14C]methyliodide (Dupont) as described (32Sasagawa T. Titani K. Walsch K.A. Anal. Biochem. 1983; 128: 371-376Crossref PubMed Scopus (14) Google Scholar). The labeled peptides were purified on reversed-phase high pressure liquid chromatography, lyophilized, and dissolved in phosphate buffer, pH 7.5. Labeled P3 had a specific activity of 8000 cpm/nmol. A competition assay between labeled and non-labeled P3 proved that both bind actin with the same affinity (data not shown). We followed actin polymerization by measuring the increase in fluorescence of pyrene-labeled actin (10%) on a SFM 25 fluorometer (Kontron Instruments, Zurich) using 365 nm as excitation and 388 nm as emission wavelength. G-actin (12 μm) in G-buffer was induced to polymerize by addition of 100 mm KCl and 1 mm MgCl2 at time 0. When analyzing the effect of the cofilin peptides, we added them in a molar excess over actin ranging from 25- to 50-fold, together with the salt at the start of the measurement. The samples were monitored during 20 min, in which the control sample (12 μm actin without cofilin peptides) reaches the fluorescence associated with steady state. A G-actin solution was dialyzed overnight against phosphate buffer (5 mm potassium phosphate, pH 7.5, 0.2 mm CaCl2, 0.2 mm ATP, 0.2 mm dithiothreitol). We added the14C-labeled cofilin peptide P3 in various molar ratios to prepolymerized actin (12 μm) (see Fig. 2 b) and incubated these samples for 30 min at room temperature and 2 h at 4 °C prior to adding the zero-length cross-linker 1-ethyl-3(3-dimethylamino-propyl)carbodiimide (EDC) (Sigma) and N-hydroxysulfosuccinimide (Pierce) to a final concentration of 4 mm each (33Staros J.V. Wright R.W. Suringle D.M. Anal. Biochem. 1986; 156: 220-222Crossref PubMed Scopus (772) Google Scholar). The reaction was kept at room temperature for another 45 min. We analyzed aliquots of the samples on SDS-PAGE mini slab gels which were stained with Coomassie Blue and quantified the degree of cross-linking using a PhosphorImager (Molecular Dynamics) and the ImageQuant software package. We performed measurements on a Jasco J-710 spectropolarimeter scanning from 184 to 200 nm with a step resolution of 0.5 nm. The peptides, at a concentration of 40 μm, were in 10 mm sodium phosphate, pH 7.5, and 3,3,3-trifluoroethanol (TFE, 60%) and at 20 °C. The data are the average of nine scans and are expressed as θ MRW (mean residue weight elipticity) as a function of wavelength. G-actin (initial concentration >12 μm) was allowed to polymerize in 10 mm Pipes, pH 6.8, 0.2 mm dithiothreitol, 0.2 mm ATP, 0.1 mm CaCl2 containing 100 mm KCl and 1 mm MgCl2. The final actin concentration after addition of all components will be 5 μm. Next we added S2–3 and cofilin (in the same buffer as actin) in such a way that the sum of both was 10 μm throughout the whole series, but their molar ratio varied, with 10:0 and 0:10 forming the two extremes (see Fig. 4, a and b). This 2-fold molar excess over actin is, for both S2–3 and cofilin separately, sufficient to saturate F-actin. The samples were kept at 4 °C overnight after a 30-min incubation at room temperature. F-actin and its associated proteins were subsequently spun down in an Beckman Airfuge at 30 p.s.i. (100,000 × g) for 30 min at room temperature. Supernatant and resuspended pellet were analyzed on SDS-mini slab gels followed by Coomassie Blue staining. We used densitometry to quantify the results. From the equilibrium dissociation constant of the actin-cofilin (K c) and actin-S2–3 complex (K s) we can derive equations (Equations 1 and 2) that render a relation between either the amount of actin-cofilin (AC) or actin-S2–3 (AS) formed as a function of the total concentration of cofilin (C tot). Ctot=(5+5n)(AC)+(1−n)(AC)25n−(n−1)(AC)Equation 1 and Ctot=(1−n)(AS)2+(5n−15)(AS)+505+(n−1)(AS)Equation 2 This takes into account that the sum of the total added concentration of cofilin and S2–3 is always 10 μm, and we assume that the sum of the amount of actin-cofilin (AC) and actin-S2–3 (AS) complex is constant in all samples as the filament is saturated (at two out of 12 data points (C tot = 6 and 7 μm) this may not be the case). This sum thus equals 5 μm (this is not taking into account the critical monomer concentration for actin polymerization and the limited extent of depolymerization induced by cofilin at pH 6.8 (which maximally amounts to about 25%)). n is the ratio of the two equilibrium dissociation constants K s /K c. Using these equations, curves can be drawn for all possible n values, and the one fitting our experimental data has an n value between 2.5 and 3, which implicates that under our conditions (and with the introduced assumptions) K s is estimated to be 2.5- to 3-fold K c which is in the range of reported K d values for binding of actin to S2–3 (ranging from 0.2–2 μm for S2–3 (F-actin)) and to cofilin (0.2–0.9 μm (G-actin)) (34Way M. Pope B. Weeds A.G. J. Cell Biol. 1992; 119: 835-838Crossref PubMed Scopus (126) Google Scholar, 35Yin H.L. Iida K. Janmey P.A. J. Cell Biol. 1988; 106: 805-812Crossref PubMed Scopus (105) Google Scholar, 36Muneyuki E. Nishida E. Sutoh K. Sakai H. J. Biochem. (Tokyo). 1985; 97: 563-568Crossref PubMed Scopus (25) Google Scholar). We added the first actin-binding protein or peptide in a constant amount to several samples of polymerized actin in phosphate buffer (see above) and incubated them for 1 h at room temperature. At this time the "competitive" peptide or protein was added in increasing concentrations to the different samples. After incubation to allow complex formation (overnight at 4 °C), we added the cross-linker EDC and N-hydroxysulfosuccinimide and let them react for another 45 min at room temperature. SDS gels were run for analysis. To assay the competition between PS2 and P3 for cross-linking to F-actin, we used PS2 labeled with 14C on the NH2-terminal cysteine. PS2 was added in a range of 3–250 μm to 12 μm polymerized actin preincubated with either 150 μm P3 or without P3. Competition between PS2 and PS1 was studied in the same way. Conversely, F-actin (12 μm) was first incubated with labeled PS2 (using either 20, 40, or 100 μm), and subsequently, P3 was added (range 0–200 μm). In both setups, the yield of labeled PS2 cross-linking to actin was quantified using a PhosphorImager (Molecular Dynamics) and the ImageQuant software package. The rabbit polyclonal anti-actin antibody against the COOH terminus was produced by the Centre d'Economie rurale, Laboratoire d'Hormonologie (Marloie, Belgium). A chemically synthesized actin peptide (residues 354–375) coupled to keyhole limpet hemocyanin following the procedure of Mumby and Gilman (37Mumby S.M. Gilman A.G. Methods Enzymol. 1991; 195: 215-253Crossref PubMed Scopus (102) Google Scholar) was used as antigen. The monoclonal α-sarcomeric actin-specific antibody was obtained from Sigma. Western blots were performed as described (38Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar). We added 120 μm of the cofilin wild type peptide P3, whereas 10 μm of cofilin was added to 5 μmF-actin. We analyzed the actin binding capacity of a peptide that combines two regions of cofilin which have previously been implicated in actin binding. This peptide (P3, Fig.1) spans residues 102–130 of the pig cofilin sequence and contains the LKSKM motif (studied in Ref. 22Yonezawa N. Nishida E. Iida K. Kumagai H. Yahara I. Sakai H. J. Biol. Chem. 1991; 266: 10485-10489Abstract Full Text PDF PubMed Google Scholar) and the more COOH-terminally located DAIKKKL motif (studied in Ref. 23Yonezawa N. Nishida E. Ohba M. Seki M. Kumagai H. Sakai H. Eur. J. Biochem. 1989; 183: 235-238Crossref PubMed Scopus (44) Google Scholar). The shorter peptides P1 (residues 104–115) and P2 (residues 118–130) each containing one motif served as control (Fig. 1). Using a similar assay as described in Ref. 22Yonezawa N. Nishida E. Iida K. Kumagai H. Yahara I. Sakai H. J. Biol. Chem. 1991; 266: 10485-10489Abstract Full Text PDF PubMed Google Scholar, we first examined the inhibitory effect of these peptides on salt-induced actin polymerization by following the fluorescence increase accompanying the polymerization of pyrene-labeled actin (10% labeled). Although intact cofilin appeared to quench fluorescence of the pyrene label (39Nishida E. Maekawa E. Muneyuki E. Sakai H. J. Biochem. (Tokyo). 1984; 95: 387-398Crossref PubMed Scopus (38) Google Scholar), we and others (22Yonezawa N. Nishida E. Iida K. Kumagai H. Yahara I. Sakai H. J. Biol. Chem. 1991; 266: 10485-10489Abstract Full Text PDF PubMed Google Scholar) did not observe this for the actin binding peptide. Fig. 2 a shows that P3 slows down actin polymerization in a concentration-dependent fashion as was also shown for P1 (22Yonezawa N. Nishida E. Iida K. Kumagai H. Yahara I. Sakai H. J. Biol. Chem. 1991; 266: 10485-10489Abstract Full Text PDF PubMed Google Scholar). However, peptide P3 is more active compared with the control peptides P1 and P2, indicating that this long peptide binds actin monomers more efficiently. This indicates that both motifs cooperate in the actin interaction. Adding a 50-fold molar excess of P3 (i.e. 600 μm) over actin at the start of polymerization results in only 14.5 and 65.5% of the F-actin formed at the half-time and at the time of complete polymerization in the control sample, respectively (Fig.2 a). In addition, using the zero-length cross-linker EDC, we show that the wild type peptide P3 can be covalently coupled to actin under polymerizing conditions. Due to the small size of the peptide the upward shift on SDS-PAGE of the actin-peptide cross-linked complex relative to the non-cross-linked actin is hardly visible. For this reason, we prepared a radiolabeled version of the cofilin peptide in which Met115 carries a 14C. This allows a quantification of the yield of cross-linking of P3 to actin as a function of peptide concentration. Fig. 2 b shows that at an actin concentration of 12 μm, saturation is reached around 60 μm of P3, supporting the specificity of binding by the cofilin peptide. From the three-dimensional structure of the mammalian cofilin homolog destrin (24Hatanaka H. Ogura K. Moriyama K. Ichikawa S. Yahara I. Inagaka F. Cell. 1996; 85: 1047-1055Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and of yeast cofilin (25Fedorov A.A. Lappalainen P. Fedorov E.V. Drubin D.G. Almo S.C. Nat. Struct. Biol. 1997; 4: 366-369Crossref PubMed Scopus (94) Google Scholar), it is evident that the sequence corresponding to P3 contains a kinked α-helix (residues 112–128). Consequently, when the peptide binds to actin it must become α-helical. Using circular dichroism, we show that P3 is capable of adopting an α-helical conformation (Fig.3). This structure is not stable in aqueous solution but becomes evident upon addition of 60% 3,3,3-trifluoroethanol (TFE), a known α-helix stabilizing agent (40Nelson J.W. Kallenbach N.R. Proteins Struct. Funct. Genet. 1986; 1: 211-217Crossref PubMed Scopus (402) Google Scholar). In contrast, P1 does not adopt an α-helical conformation under these conditions, and the shorter peptide P2 is also less helical than P3. The structural homology between destrin and segments of gelsolin, as demonstrated by Hatanaka and co-workers (24Hatanaka H. Ogura K. Moriyama K. Ichikawa S. Yahara I. Inagaka F. Cell. 1996; 85: 1047-1055Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), evidently raises the question whether there is any functional homology between these two families of actin-modulating proteins. Members of the gelsolin family are able to sever and/or cap actin filaments. The multiple domains of gelsolin cooperate to obtain this double effect, with segment 1 + 2 being the minimal efficient severing portion. It is well documented that F-actin binding by segment 2 (S2) is a prerequisite for severing (41Weeds A.G. Maciver S. Curr. Opin. Cell Biol. 1993; 119: 835-842Google Scholar). Cofilin and its homologs were also proposed to possess weak severing activity (15Hayden S.M. Miller P.S. Brauweiler A. Bamburg J.R. Biochemistry. 1993; 32: 9994-10004Crossref PubMed Scopus (202) Google Scholar, 42Hawkins M. Pope B. Maciver S.K. Weeds A.G. Biochemistry. 1993; 32: 9985-9993Crossref PubMed Scopus (239) Google Scholar, 43Maciver S.K. Zot H.G. Pollard T.D. J. Cell Biol. 1991; 115: 1611-1620Crossref PubMed Scopus (192) Google Scholar), although recent reports question the severing activity and convincingly promote the idea that the interaction of cofilin rather affects the rate of treadmilling (9Carlier M.-F. Santolini J. Laurent V. Didry D. Yan H. Chua N.-H. Pantaloni D. Mol. Biol. Cell. 1996; 7: 546aGoogle Scholar). However, whatever mechanism is used by cofilin, an initial contact with F-actin will be a first and essential step, and for the purpose of this paper we will consider this initial F-actin binding as a "function" regardless of its effect on actin dynamics. Consequently, we were interested in determining whether this F-actin binding property of proteins of the cofilin/ADF and gelsolin families bears any resemblance. To investigate this, we isolated the F-actin binding part of human plasma gelsolin comprising segment 2 and 3 (S2–3) and performed competition experiments under conditions (pH 6.8) where cofilin is known to efficiently bind to F-actin and only causes limited depolymerization (14Yonezawa N. Nishida E. Sakai H. J. Biol. Chem. 1985; 260: 14410-14412Abstract Full Text PDF PubMed Google Scholar). Earlier studies have shown that S2–3 and cofilin can both form a 1:1 complex with actin protomers in the filament (36Muneyuki E. Nishida E. Sutoh K. Sakai H. J. Biochem. (Tokyo). 1985; 97: 563-568Crossref PubMed Scopus (25) Google Scholar, 41Weeds A.G. Maciver S. Curr. Opin. Cell Biol. 1993; 119: 835-842Google Scholar). It is generally accepted that in S2–3, S2 contains the F-actin binding site and S3 is not contributing directly to F-actin binding (see also "Discussion"). Fig. 4 a(dashed lines) shows the densitometric analysis of a sedimentation assay in which binding to F-actin was analyzed for cofilin or S2–3, separately. It shows that at an actin concentration of 5 μm saturation of binding to F-actin protomers is reached for both S2–3 and cofilin at an added concentration of 5 and 7.5 μm, respectively. We tested competition between S2–3 and cofilin for binding to F-actin using a continuous variation experiment. Fig. 4 b shows the composition of the pellets after high speed centrifugation of a series of samples in which the molar ratio of S2–3 and cofilin varies, but their total molarity (S2–3 + cofilin) remains constant at a 2-fold molar excess over actin. The amount of each complex formed (either actin-cofilin or actin-S2–3) is determined from the amount of cofilin and S2–3 in the pellet after high speed centrifugation and plotted in Fig. 4 a(solid lines) as a function of total cofilin concentration. The fact that, in the presence of cofilin, the amount of S2–3 bound to F-actin is lower, compared with samples containing equal concentrations of S2–3 but no cofilin (and vice versa), and that the reduction in the F-actin bound S2–3 is compensated by an increase in bound cofilin of about similar size indicates that cofilin and S2–3 displace each other and therefore suggests that both proteins compete for the same or an overlapping site on F-actin. In addition, densitometric scanning of cross-linking experiments shows that a saturable amount of the actin-binding peptide of cofilin (250 μm P3) decreases the yield of cofilin binding to F-actin (Fig. 4 c). The same effect was observed for the binding of cofilin in the presence of the segment 2 gelsolin peptide PS2. This chemically synthesized peptide contains part of the F-actin binding site of gelsolin segment 2 (residues 198–227, see Fig. 1), competes with S2–3 for binding to F-actin, and slo
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