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* Institute of Dental Sciences, School of Dental Medicine, Department of Pharmacology, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel; and Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California
Correspondence: Address reprint requests to Andras Muhlrad, Fax: 972-2-675-8561; E-mail: muhlrad{at}cc.huji.ac.il.
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ABSTRACT |
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-ADP upon cofilin binding and a decrease in its accessibility to collisional quenchers. BeFx also affects the nucleotide-binding cleft of F-actin, as indicated by an increase in the fluorescence intensity of -ADP-F-actin. Phalloidin and cofilin inhibit, but do not exclude each other binding to their complexes with F-actin. Phalloidin promotes the dissociation of cofilin from F-actin and slowly reverses the cofilin-induced disorder in the DNase I binding loop of subdomain 2. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Phalloidin, and inorganic phosphate (Pi) and its analogs—beryllium fluoride (BeFx) and aluminum fluoride (AlF4)—belong to the group of F-actin stabilizing factors. These small molecules stabilize F-actin by reducing the critical concentration for polymerization and introducing conformational changes into the filament structure.
The complexes of beryllium and aluminum with fluoride were found to be good structural analogs of Pi (1
) and are widely used in studying the activity of various nucleotide binding proteins, including G-proteins, Na+, K+-ATPase, tubulin, and others (2). Combeau and Carlier found that BeFx (BeFx stands for the 
and BeF2(OH)– complexes) and AlF4 bind strongly to F-actin (3). BeFx and AlF4 bind to F-actin with orders of magnitude higher affinity (Kd = 2 µM and 25 µM, respectively) than Pi (Kd = 1.5 mM). BeFx competes with Pi for binding to the nucleotide-binding cleft of ADP-F-actin protomers at the place of the 
-phosphate of ATP (3). It stabilizes strongly F-actin by decreasing the rate of protomer dissociation (
150-fold) and the critical concentration for polymerization (100-fold) (3). BeFx stabilizes in particular the structure of subdomain 2, as indicated by strong and cooperative inhibition of its cleavage by subtilisin (in the DNase I binding-loop (D-loop)) and trypsin (in the 60–69 loop) (4), and by electron microscopy studies (5). These effects of BeFx on F-actin are similar to those of Pi (3,4), but BeFx is more effective at much smaller concentrations. BeFx-induced changes in the C-terminus region were also detected by fluorescence (3) and proteolysis (4) methods.
Phalloidin has the strongest stabilizing effect on actin filaments. It decreases the critical concentration of actin polymerization, reduces the rate of monomer dissociation from both filament ends (6), and inhibits phosphate release from the nucleotide binding cleft after ATP hydrolysis. Phalloidin binds at the interface of three actin monomers (7–9) and stabilizes lateral interactions between the two filament strands.
Among actin destabilizing factors, the actin depolymerizing factor/cofilin (ADF)/cofilin) family of proteins, or AC proteins (10), have attracted much attention because of their important role in regulating actin dynamics in cells. These proteins change the twist of actin filaments (11), and destabilize, sever (12), and depolymerize (13) them by weakening longitudinal (14,15) and lateral (16,17) interprotomer contacts in F-actin. Extensive, cofilin-induced conformational changes in subdomain 2 of F-actin are readily monitored via quenching of the fluorescence of tetramethyl rhodamine cadaverine (TRC) (attached to Gln-41 on the D-loop (18)), and a strong acceleration of subtilisin (between Met-47 and Gly-48) and tryptic cleavage (after Arg-62 and Lys-68) in subdomain 2 (18).
The antagonistic structural effects of AC proteins and BeFx (and phalloidin) on F-actin appear consistent with their reciprocal inhibition of binding to F-actin (13) and the reported blocking of Acanthamoeba actophorin (AC protein) binding by BeFx (19) and human cofilin binding by phalloidin (20) to F-actin. On the other hand, we found that yeast cofilin removes rhodamine-phalloidin from F-actin (17), and obtained preliminary evidence (21) that BeFx inhibits the rate but not the extent of yeast cofilin binding to muscle F-actin. Following these observations, we used here the binding of yeast cofilin to BeFx-F-actin as a tool to study the structural effects of BeFx on actin filaments. Cofilin binding to F-actin was monitored via changes in the susceptibility of subdomain 2 to subtilisin and trypsin, and via changes in the fluorescence intensity of the TRC probe attached Gln-41 (18) and of 1,N6-ethenoadenosine diphosphate (-ADP) bound in the nucleotide-binding cleft of F-actin. In addition, we also monitored the effect of cofilin on the release of BeFx from F-actin by 19F-NMR. We found that BeFx strongly inhibits the rate, but not the extent of cofilin binding, while cofilin greatly facilitates the dissociation of BeFx from F-actin, and affects the structure of subdomain 2 and the nucleotide binding cleft of F-actin. Our results indicate the existence of two types of BeFx-F-actin complexes, with different conformations and different rates of cofilin binding.
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MATERIALS AND METHODS |
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-ATP) trypsin, soybean trypsin inhibitor, subtilisin (Carlsberg), phalloidin and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical (St Louis, MO). Bacterial transglutaminase was a generous gift from Dr. K. Seguro (Ajimoto, Kawasaki, Japan).
Proteins
G-actin was prepared from back and leg muscles of rabbit by the method of Spudich and Watt (22) and stored in G-buffer containing 5.0 mM TrisHCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM dithiotreitol, pH 8.0. F-actin was prepared from G-actin by polymerizing it with 2.0 mM MgCl2. Recombinant yeast cofilin was prepared as described before (23) with minor modifications (15). The concentrations of cofilin and unlabeled skeletal muscle 
-actin were determined spectrophotometrically by using the extinction coefficients 
and 
, respectively. (The optical density of actin was measured in the presence of 0.5 M NaOH, which shifts the maximum of absorbance from 280 to 290 nm). Molecular masses were assumed to be 42 and 15.9 kDa for skeletal actin and yeast cofilin, respectively.
Proteolysis
Labeled or unlabeled F-actin (10 µM) was digested in the presence and absence of cofilin in pH 8.0 F-buffer (20.0 mM TrisHCl, 2.0 mM MgCl2, 0.2 mM ATP, 0.5 mM dithiotreitol), and pH 6.5 F-buffer (20.0 mM PIPES, 2.0 mM MgCl2, 0.2 mM ATP, 0.5 mM dithiotreitol), with 25 µg/ml subtilisin or 800 µg/ml trypsin, respectively. The products of digestion were run on SDS-PAGE. Protein bands on SDS gels were analyzed by densitometry.
Chemical modification
Actin labeled with TRC at Gln-41 (TRC-actin) was prepared by incubating 50 µM skeletal G-actin with 100 µM TRC and 0.18 mg/ml bacterial transglutaminase in G-buffer pH 8.0, at 22°C for 2 h. Reagent excess was removed by filtering actin through a PD-10 column equilibrated with G-buffer. The extent of actin labeling for TRC was estimated using extinction coefficient of E554 = 78,000 cm–1M–1. The concentration of the labeled actin was measured by the Bradford protein assay (24), using native actin as a standard.
Preparation of -ADP-F-actin
This was done essentially as described previously (25). Briefly, skeletal muscle G-actin was passed through a desalting column (Amersham, PD10, Piscataway, NJ) of Sephadex G-25 equilibrated with ATP-free G-buffer. The eluted actin was supplemented with a 20-fold molar excess of -ATP and was incubated for 1 h on ice. Excess -ATP was removed from G-actin by passing it through another PD10 column. Actin was polymerized by addition of 2.0 mM MgCl2 and during the polymerization the actin-bound -ATP was hydrolyzed to -ADP.
Fluorescence measurements
Fluorescence measurements were carried out at 22°C with a PTI spectrofluorometer (Photon Technology Industries, South Brunswick, NJ) in pH 8.0 and pH 6.5 F-buffer. For TRC and -ADP, the excitation wavelength was set at 544 and 350 nm and the emission at 580 and 412 nm, respectively. Emission spectrum of -ADP-F-actin was recorded between 370 and 550 nm wavelengths.
19F NMR measurements
Spectra were obtained on the Varian Inova 500 instrument at 470.215 MHz, in a 5-mm probe. Spectrum width was 50,000 Hz, delay time 1 s, and acquisition time 0.7 s. Line broadening function was set to 20 Hz. In all experiments temperature was kept at 20°C and the solutions were supplemented with 15% D2O.
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RESULTS |
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). Here, we monitored the decrease in TRC fluorescence (Fig. 1) upon addition of 12 µM cofilin to 10 µM TRC-F-actin in the presence and absence of 60 µM BeFx at pH 8.0 and 6.5 (BeFx alone did not affect the fluorescence of TRC-F-actin). In the absence of BeFx, the decrease in the fluorescence of TRC-F-actin by cofilin was essentially completed within the mixing time of the solutions, in agreement with our earlier observations (18). Yeast cofilin also binds to TRC-F-actin in the presence of BeFx at both pH-s, but at a much slower rate than in its absence. Fig. 1 shows that BeFx inhibits the rate, but not the extent of cofilin binding and that this binding is faster at pH 6.5 than at pH 8.0. The time course of fluorescence changes could be well fitted to a two-exponential expression, yielding the apparent first order fast (kf) and slow (ks) cofilin binding rates at pH 6.5 and 8.0, from which the second order association rate constants were calculated by taking into account their dependence on cofilin concentration (Fig. 2 and Table 1).
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,19), we have confirmed the binding of yeast cofilin to BeFx-TRC-F-actin also by their cosedimentation at pH 8.0 (Fig. 3). Similar results were obtained also with unlabeled actin, both at pH 6.5 and 8.0 (data not presented), showing that yeast cofilin binds to F-actin in the presence of BeFx.
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). Cofilin binding has the opposite effect, as it increases dramatically the proteolysis of the D- and 60–69-loops by subtilisin and trypsin (18). We show in Fig. 4 A that cofilin increases the rate and extent of subtilisin cut in the D-loop of BeFx-F-actin at pH 6.5 and pH 8.0, with a bigger effect noted at the lower pH. This is consistent with the TRC-F-actin fluorescence results (Fig. 1). The rate and the extent of subtilisin cleavage increased with the time of cofilin incubation with BeFx-F-actin (Fig. 4 B). Similar results were obtained also for the tryptic cleavage of the 60–69 loop (after Arg-62 and Lys-68), which became faster and more extensive with cofilin incubation (Fig. 4 C). It should be noted, however, that even after 20 min incubation with cofilin both the subtilisin and trypsin digestions were less extensive than in the presence of cofilin without BeFx. The results of proteolysis experiments confirm cofilin binding to BeFx-F-actin.
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80% of the bound fluoride dissociates from F-actin upon 30 min incubation with cofilin. These results indicate that the binding of cofilin induces conformational changes in the nucleotide-binding cleft of F-actin, and decreases BeFx affinity to actin.
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-ADP analog. We found that upon addition of BeFx to -ADP-F-actin the fluorescence intensity of the bound -ADP increases by 11% (Fig. 6 A). The time course of the binding of BeFx to -ADP-F-actin (Fig. 6 B), which was fitted to a single exponential expression, is rather slow. The calculated second order association rate constant of this reaction is 3.67 ± 0.57 x 10–5 s–1 µM–1. BeFx does not affect the quenching of the fluorescence intensity of actin bound -ADP by nitromethane (Fig. 6 C), (KSV values at pH 8.0 in the absence and presence of BeFx are 1.87 ± 0.1 M–1 and 1.86 ± 0.1 M–1, respectively). On the other hand cofilin significantly reduces the accessibility of nitromethane to -ADP in the nucleotide-binding cleft of F-actin (KSV = 0.926 ± 0.04 M–1) (see also Muhlrad et al. (25)). Addition of cofilin to -ADP-F-actin also increases the fluorescence intensity of the bound -ADP by 50% (25), which is significantly more than the BeFx induced increase (Fig. 6 A). The fluorescence change observed upon adding cofilin in the absence of BeFx to -ADP-F-actin is very fast while it is slow in the presence of BeFx (Fig. 6 B), which is consistent with TRC-F-actin fluorescence results (Fig. 1). When cofilin was added to BeFx--ADP-F-actin the overall fluorescence increase was smaller (30%) than that with cofilin and BeFx-free -ADP-F-actin (50%) (Fig. 6).
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1:2 mol ratio of cofilin/actin. Predictably, both the relative extent and rate of the fast and slow fluorescence decrease upon cofilin binding to TRC-F-actin depended on BeFx concentration (up to 60 µM), as did also the tryptic digestion of such actin (data not shown).
More revealing were the experiments in which we examined the effect of F-actin preincubation with BeFx on the binding of cofilin (Fig. 7 A). TRC-F-actin (10 µM) was incubated with 60 µM BeFx for 1 and 24 h, respectively, and then mixed with cofilin (5.6 µM). The initial, fast fluorescence decrease was faster and greater for the sample incubated with BeFx for 1 h than for 24 h. Similarly, the rate and the extent of subtilisin digestion of BeFx-F-actin in the presence of cofilin were greater after 1 h than 24 h incubation with BeFx (Fig. 7 B). These results indicate that the binding of cofilin to BeFx-F-actin depends on the incubation time of F-actin with this phosphate analog, i.e., most likely, on the ratio of the strongly to the weakly bound BeFx-F-actin complex, which increases with the time of incubation. Interestingly, in the absence of cofilin no difference was observed in the inhibition of subdomain 2 proteolysis between samples incubated for 1 h and 24 h with BeFx. In fact, full protection against proteolysis of F-actin by subtilisin appears already after 5 min incubation with 60 µM BeFx (Fig. 7 C). This result is consistent with the time course of the fluorescence intensity increase observed upon adding BeFx to -ADP-F-actin (Fig. 6 B). Similar results were obtained at pH 6.5 with subtilisin and at pH 8.0 with trypsin digestion (data not shown). Thus, although both the weakly and strongly bound BeFx-F-actin complexes are equally well protected against proteolysis in the absence of cofilin, they appear to present different binding environments to cofilin.
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). Here we tested the effect of phalloidin on cofilin binding to F-actin by subtilisin digestion, taking advantage of the strong phalloidin inhibition of the D-loop cleavage (26). Phalloidin, 12 µM, was added to 10 µM F-actin containing 5 or 12 µM cofilin and after 1.5 or 22 h incubation at pH 8.0 the samples were digested with subtilisin. We also reversed the order of additions, adding phalloidin first and cofilin second. The results of such digestions in the presence of 12 µM cofilin are presented in Fig. 9. The digestion pattern clearly shows that the addition of phalloidin to cofilin-F-actin decreases, whereas the addition of cofilin to phalloidin F-actin increases the rate and extent of subtilisin cleavage. However, the extent of actin cleavage after 90 min incubation (Fig. 9 A) was greater when phalloidin was added to cofilin-F-actin than in the case of a reversed order of additions, when cofilin was added to phalloidin-F-actin. Such digestion differences disappeared after 22 h incubation (Fig. 9 B), showing the slow equilibration of this system. These results indicate that phalloidin, unlike BeFx or inorganic phosphate (25), binds to F-actin also in the presence of cofilin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) binds strongly to F-actin, stabilizing its structure, and according to several reports prevents the binding of some ADF/cofilin proteins (Acanthamoeba actophorin (19), human cofilin (20), and plant ADF (13)) to F-actin. We found in this study that in contrast to above-mentioned three members of the AC family, yeast cofilin binds to BeFx-F-actin, albeit at a much slower rate than to F-actin. This feature of yeast cofilin indicates that it has a higher affinity to F-actin than those AC family members that do not bind to F-actin in the presence of BeFx. The high affinity of yeast cofilin to F-actin may have physiological significance. We took advantage of this property of yeast cofilin and examined its binding to BeFx-actin to gain further insight into the changes caused in actin structure by this phosphate analog.
In agreement with Ressad et al. (27), we found that the binding of cofilin is faster at low than at high pH (25), although the depolymerizing effect is stronger at the high pH (28). In the presence of BeFx the binding rates at pH 8.0 and pH 6.5 are much closer (Table 1). It is possible that the nature of the beryllium fluoride complex that binds to F-actin contributes to the change in the rate difference at the two pH values. According to Combeau and Carlier (29), at low pH only binds, whereas at high pH both and BeF2(OH)– bind to F-actin (only ions with a single negative charge bind to the nucleotide-binding cleft of actin). may bind stronger to F-actin than BeF2(OH)– thereby, inhibits more effectively cofilin binding, which would explain the relatively stronger inhibition of cofilin binding at low pH. Similar conclusion was reached by studying the activation of transducin by the above two beryllofluoride complexes (30).
We monitored the binding of cofilin to F-actin by following the decrease in fluorescence intensity of the TRC group attached to Gln-41. The rate of cofilin binding was found to increase with increasing cofilin concentration without reaching a plateau even at 10:1 cofilin/actin molar ratio. This indicates that cofilin binds to BeFx containing F-actin protomers and the BeFx dissociation does not limit the rate of cofilin binding. This conclusion is supported by the results of Combeau and Carlier (3), who found by two methods (rapid dialysis and chasing out 7Be with unlabeled Be) that the rate of dissociation of BeFx from F-actin is extremely slow 10–6 s–1. We suggest that cofilin binding to BeFx containing protomers causes conformational changes at the BeFx binding site in the nucleotide-binding cleft. These changes induce dramatic decrease in the affinity of BeFx to F-actin, leading to its dissociation.
We studied the effects of BeFx and cofilin on the nucleotide-binding cleft of F-actin also by detecting changes in the fluorescence of -ADP bound to F-actin. The different effects of these two ligands on -ADP fluorescence and collisional quenching indicate that the structural changes induced by the two ligands in the cleft are different. The BeFx induced -ADP fluorescence intensity increase is consistent with the proposal of Combeau and Carlier (3,29) that BeFx is located at the place of -phosphate of ATP in the nucleotide-binding cleft of actin. The incomplete fluorescence intensity increase by cofilin in the presence of BeFx is presumably due to the residual bound BeFx inhibiting cofilin's effect on the conformation of the nucleotide-binding cleft. This finding is consistent with the effect of cofilin on the extent of proteolysis of the subdomain 2 of F-actin in the presence and absence of BeFx.
The binding of cofilin to F-actin in the presence of BeFx was also monitored by the increase in proteolytic susceptibility of subdomain 2. Since cofilin increases while BeFx decreases proteolytic susceptibility, this method also measures BeFx dissociation. The results of proteolytic experiments show that the dissociation of BeFx is not complete even 20 min after the addition of cofilin (Fig. 4, B and C), when according to the fluorescence measurements F-actin is fully saturated with cofilin. This indicates that after 20 min incubation with cofilin there are actin protomers to which BeFx and cofilin are simultaneously bound. The F-actin-bound residual BeFx inhibits considerably the proteolysis of subdomain 2 due to its strong cooperative effect on F-actin structure (4).
The binding of BeFx to F-actin is a relatively slow, two-step process (3). The first step is a fast equilibrium binding, which is followed by a slow isomerization step accompanied by a conformational change. We measured the rate of BeFx binding to F-actin by following the increase in the fluorescence intensity of -ADP-F-actin and found that it takes 5 min for the fluorescence to reach the plateau after addition of BeFx (Fig. 6 B). About the same time is needed for F-actin to become fully resistant to proteolysis upon addition of BeFx (Fig. 7 C). However, cofilin binding experiments revealed that further changes occur in the structure of BeFx-F-actin, even after it became resistant to proteolysis. We showed that the binding of cofilin to BeFx-F-actin is also a two-step process consisting of a fast and a slow step. The amplitude of the fast step decreases with the time of incubation with BeFx; it is much smaller after 24 h than 1 h incubation with BeFx (Fig. 7 A). The ratio of the amplitudes of the fast and slow steps also depends on BeFx concentration; with the relative size of the first step decreasing with increasing BeFx concentration. We associate the fast and slow steps with the low and high affinity BeFx-F-actin complex, respectively. Since both complexes are resistant to proteolysis and their -ADP fluorescence is increased, we conclude that cofilin binding detects a second isomerization step in the BeFx-F-actin interaction, according to Scheme 1:
 | (Scheme 1) |
BeFx-F-actin is the fast equilibrium complex and BeFx-F-actin* and BeFx-F-actin** are the low- and high-affinity BeFx-F-actin complexes, respectively. A structural difference between the two complexes is suggested by the different degree of inhibition of cofilin binding. It appears that the same beryllium fluoride species binds to F-actin in the various BeFx-F-actin complexes, because the transformations between these complexes are not pH dependent.
It is interesting to compare the BeFx-F-actin complexes with the complexes of Pi-F-actin. According to Combeau and Carlier (3) there are two ADP-Pi-F actin complexes. Pi dissociates slowly from the ADP-Pi-F-actin* complex, which is a product of ATP hydrolysis accompanying actin polymerization, while it dissociates fast from the ADP-Pi-F actin complex, which is produced upon addition of Pi to ADP-F-actin. The two complexes are connected by an isomerization step, which limits the transformation of ADP-Pi-F-actin* to ADP-Pi-F actin according to Scheme 2.
 | (Scheme 2) |
The reversal of the isomerization step is extremely slow and thus, essentially only the ADP-Pi-F actin complex is obtained upon adding Pi to ADP-F-actin. We may assume that BeFx-F-actin** could be similar to the ATP-F-actin and BeFx-F-actin* to the ADP-Pi-F-actin* complex. This speculation is supported by the results of Combeau and Carlier (3) on the effect of BeFx on the fluorescence of pyrene-labeled F-actin. However, this hypothesis needs to be corroborated with further evidence.
Cofilin binding induces the dissociation of BeFx from F-actin, similarly to that of phalloidin (17). The removal of BeFx from actin indicates that cofilin causes allosteric conformational changes also in the nucleotide-binding cleft of F-actin where the BeFx is bound. Cofilin-induced conformational changes in the nucleotide cleft were indicated also by decreased phosphate affinity, changed fluorescence emission spectra, and decreased accessibility of F-actin-bound -ADP to collisional quenchers (Fig. 6 and Muhlrad et al. (25)).
BeFx cannot bind to F-actin when the filaments are fully saturated with cofilin as shown by the inability of BeFx to reverse the cofilin-induced changes in the fluorescence intensity and proteolytic susceptibility. This indicates lower probability for the initial complex formation (BeFx-F-actin) and its reduced isomerization when the nucleotide-binding cleft of F-actin is allosterically changed by cofilin (Scheme 1). Cofilin probably inhibits the first isomerization step of BeFx, as the proteolytic susceptibility of the F-actin-cofilin complex remains high after the addition of BeFx. The effect of cofilin on BeFx binding is not cooperative; because the binding appears to be prevented only in those protomers of F-actin that are saturated with cofilin.
Phalloidin, unlike BeFx, can bind to cofilin-F-actin, which is fully saturated with cofilin. It competes with cofilin for F-actin and very slowly reverses the cofilin-induced disorder in the D-loop of subdomain 2 of F-actin. The different effect of BeFx and phalloidin on cofilin-F-actin is probably due to their binding to different sites on F-actin (3,7,8), and to the higher affinity of phalloidin to F-actin: (Kd = 2.1 nM; (31)) than BeFx (Kd = 2 µM; (3)) to F-actin.
In contrast to Acanthamoeba actophorin, plant ADF, and human cofilin (19,13,20), yeast cofilin not only binds to F-actin in the presence of phalloidin or BeFx, but also facilitates their dissociation (Fig. 5; (17)). The binding of cofilin to BeFx-F-actin revealed the existence of two types of BeFx-F-actin complexes, which transform to each other. The transformation of the weakly to the strongly bound complex is a very slow process, while the preceding step, of the initial complex formation, depends also on BeFx concentration. It is conceivable that the transformation of the complexes is accompanied by the movement of BeFx in the nucleotide binding cleft, which affects the conformation of the cleft and allosterically induces changes in the stability of the actin filament.
Overall, our studies resulted in several findings. By taking advantage of the high affinity of yeast cofilin to F-actin, which enables this cofilin to bind to F-actin also in the presence of BeFx or phalloidin, the antagonistic effects of the above factors were described quantitatively in this study. The existence of two BeFx-complexes with different stabilities was suggested by the pattern of cofilin binding to BeFx-F-actin. These complexes mimic different Pi-ADP-F-actin complexes, which exist during the hydrolysis of the F-actin bound ATP and upon the addition of Pi to ADP-F-actin. The study of the two BeFx-F-actin complexes may help to reveal the structure of functionally important Pi-F-actin adducts. Cofilin was found to affect the conformation of the nucleotide binding cleft of F-actin in addition to its influence on the structure of the DNase I binding loop and the 60–69 loop in subdomain 2. This effect of cofilin is manifested in the removal of BeFx from the nucleotide binding cleft, in the increase of F-actin bound -ADP fluorescence, a decrease in the accessibility of bound -ADP to collisional quencher, and the prevention of the tight BeFx binding. BeFx also increases the fluorescence intensity of the bound -ADP, which supports the earlier findings (3,29) that BeFx binds in the nucleotide cleft at the site of -phosphate of ATP. The detection of the unique effects of cofilin and the Pi analog, BeFx, on the actin structure will contribute to the understanding of the regulation of the cellular actin dynamics by AC-proteins and inorganic phosphate.
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ACKNOWLEDGEMENTS |
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Submitted on April 25, 2006; accepted for publication August 31, 2006.
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REFERENCES |
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) BeFx only; (
) BeFx and cofilin; (
) no addition; (•) cofilin only. (B) After 0.5, 5, and 20 min incubation of 10 µM BeFx-F-actin with 10 µM cofilin at 22°C, actin was digested with 18 µg/ml subtilisin at pH 8.0 for 20, 50, and 80 s. (
) BeFx only; (
) 0.5 min incubation with cofilin; (
) cofilin only, no BeFx.
) 1-h incubation with BeFx, no cofilin; (
) cofilin added first then phalloidin; (•) only cofilin added.