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J. Biol. Chem., Vol. 275, Issue 36, 28120-28127, September 8, 2000

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Actin-Latrunculin A Structure and Function

DIFFERENTIAL MODULATION OF ACTIN-BINDING PROTEIN FUNCTION BY LATRUNCULIN A^*

Elena G. Yarmola, Thayumanasamy Somasundaram, Todd A. Boring, Ilan Spector^§, and Michael R. Bubb^¶

From the Department of Medicine, University of Florida, Gainesville, Florida 32610, the  Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, the ^§ Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794, and the ^¶ Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608

Received for publication, May 18, 2000, and in revised form, June 19, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to 0.4 µM and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin ₄ to actin is inhibited by latrunculin A. Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin ₄, then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if, as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin ₄ is a surprising result that suggests that significant allosteric changes affect the thymosin ₄ binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2₁2₁2₁ and diffraction to 3.10 Å. A high resolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Latrunculin A, isolated from the Red Sea sponge Negombata magnifica, was initially identified as an inhibitor of actin polymerization by its morphological effects and by the effects it had on actin filament distribution in cultured nonmuscle cells (1). Based on the effects of latrunculin A on the steady state level of F-actin in vitro, the effects of the drug were thought to be consistent with sequestration of monomeric actin in a 1:1 molar complex with equilibrium dissociation constant of 0.2 µM (2). The binding site of latrunculin has not been conclusively identified, but based on the study of the effects of specific mutations of yeast actin on latrunculin A binding, it has been inferred that latrunculin A may bind to actin near or in its nucleotide binding cleft (3, 4). The observation that latrunculin affects nucleotide exchange has been offered as support of this conclusion (3). These data, however, are inconclusive in light of the fact that many actin-binding proteins with binding sites that are spatially distant from the nucleotide cleft are also able to affect nucleotide exchange (5) and that actin demonstrates several additional allosteric properties that serve as a precedent for the transmission of structural alterations to distant sites (6-8).

When latrunculin A is employed in studies of cell biology, the observed effects are consistent with depolymerization of actin filaments consequent to sequestration of monomeric actin by latrunculin (9). A previous preliminary report (2) did not rule out the possibility that latrunculin A has effects related to the polymerization of actin in addition to monomer sequestration, and these possibilities are explored in our current studies. Other effects of latrunculin A on the cytoskeleton are possible, however, and evidence has been reported that latrunculin can affect the expression of actin and possibly of other actin-binding proteins by a feedback mechanism that may sense the cellular concentration of actin monomers, resulting in more complicated outcomes than that predicted by monomer sequestration alone (10). To characterize the surface interactions of latrunculin A and actin, we examined whether latrunculin A affected the interaction of actin with other actin-monomer-binding proteins. To our surprise, latrunculin A inhibited binding by thymosin ₄ but not binding by profilin or DNase I. Because thymosin ₄ has been postulated to perform functions related to wound healing (11), apoptosis (12), and the inflammatory response (13), augmentation of the concentration of free thymosin ₄ by latrunculin A could potentiate these responses. Our results imply that actin-binding marine natural products may have effects other than those predicted solely by their effects on actin polymerization and, by inference, that marine natural products may exist that affect actin-binding protein function without directly affecting actin polymerization. Finally, our results illustrate a novel mechanism by which pharmacological agents that bind actin could be used to modulate the function of actin-binding proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freez, Rogers, AR) in Buffer G (5.0 mM Tris, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl₂, and 0.01% sodium azide, pH 7.8) (15), and pyrenyl-actin (actin labeled on Cys-374 with N-(1-pyrene)iodoacetamide) was prepared with 0.7-0.95 mol of label/mol of protein using the method of Kouyama and Mihashi (14). Recombinant human profilin was purified as described previously (15). Beef pancreatic DNase I (molecular biology grade; Worthington Biochemical Corp., Freehold, NJ) was reconstituted from lyophilized powder. Rat thymosin ₄ cDNA (which codes for an amino acid sequence identical to that of human thymosin ₄) in a pcDNA3 (Invitrogen, Carlsbad, CA) vector was a gift from Dr. Vivianne Nachmias (University of Pennsylvania School of Medicine). Oligonucleotides were designed so as to add a cysteine residue to the C terminus, and both strands of the cloned products were sequenced to verify the outcome. After cloning into an pET-12a expression vector, the Escherichia coli strain BL21(DE3) was transformed with plasmid. Latrunculin A was stored as a 2 or 10 mM stock in Me₂SO and was diluted to 100 µM in Buffer G for the in vitro experiments.

Purification and Labeling of Thymosin ₄-- Cells containing wild-type or cysteine-modified thymosin ₄ constructs were grown at 37 °C in M9 medium and harvested 3 h after induction with 1 mM isopropyl -D-thiogalactopyranoside. Cell pellets were dissolved in 0.5 M cooled perchloric acid, sonicated for 2 min in ice, and centrifuged for 30 min at 4 °C (130,000 × g). The supernatant was adjusted to pH 7.0-7.5 with KOH and centrifuged to remove KClO₄. After the adjustment of pH to 4.0 with formic acid, the supernatant was rapidly heated to 80 °C for 10 min, chilled on ice for 10 min, centrifuged for 30 min at 4 °C, dialyzed against 20 mM formic acid, pH 4.0, and loaded on a SP-Hi Trap column (Amersham Pharmacia Biotech). The thymosin ₄ was eluted with a linear gradient of NaCl (0-2 M) in 20 mM formic acid, pH 4.0). The fractions were neutralized with 2M Tris base as soon as eluted and dialyzed against P buffer (5 mM Tris-HCl, 40 mM KCl, 0.2 mM dithiothreitol, 0.02% sodium azide, pH 7.9). Thymosin ₄ concentration was determined using the BCA protein assay (Bio-Rad).

For labeling, thymosin ₄ was dialyzed in 50 mM sodium borate buffer, and then tetramethylrhodamine-5-maleimide (T-6027, Molecular Probes Inc.) was added in four aliquots to a final molar ratio of dye to thymosin ₄ of 2:1. After 8 h of stirring at 33-34 °C, the sample was chilled on ice and left overnight. The reaction was stopped by addition of dithiothreitol, and the sample was dialyzed against P buffer. Thymosin ₄ was then gel-filtered with Superose-12 column, and the concentration of thymosin ₄ was determined by BCA protein concentration assay. Extent of labeling was determined using extinction coefficients for dye of ₅₄₁ = 115 mM¹ and ₂₈₀ = 32.5 mM¹. Modification of the C terminus with addition of an acetylated cysteine has previously been shown not to affect the actin binding properties of thymosin ₄ (16).

Steady State and Elongation Rate Measurements-- Actin (4% pyrenyl-labeled) was converted to Mg²⁺-actin by the addition of 125 µM EGTA and 50 µM MgCl₂, and after 15 min, it was polymerized by the addition of MgCl₂ to a final concentration of 2.0 mM with varying KCl (or at 10 mM KCl and varying latrunculin A). Individual steady state samples were prepared by dilution of 10 µM F-actin without a change in buffer conditions, and steady state fluorescence readings were obtained at 24 h as described previously (17). Equilibrium dissociation constants were calculated assuming that the x intercepts reflected the total amount of unpolymerized actin, either as monomer or as a complex of latrunculin A and actin. The analysis assumes that fluorescence intensity is proportional to F-actin concentration. A seeded polymerization assay using gel-filtered cross-linked F-actin seeds was used to measure elongation rates of 4.0 µM Mg²⁺-actin as described previously (17). Preliminary data confirmed that the initial rate of polymerization was proportional to both the concentration of added seeds and to the concentration of free actin.

Nucleotide Exchange on Actin-- Excess free ATP was removed using AG 1-X8 anion exchange resin (Bio-Rad) as described previously (18). Actin (1.7 µM) and profilin (0.2 µM) were incubated in a glass cuvette with Buffer G without ATP and various concentrations of latrunculin A. A mixture of ATP and KCl (final concentrations, 3.37 µM and 50 mM, respectively) was added to start the reaction. After mixing, samples were placed in spectrofluorometer, and the time course of fluorescence changes was recorded (15). Exchange rates were obtained by fitting the time course to a single exponential. Data were then fit to the following equilibrium dissociation constants: K_dP, for profilin to actin, K_dL for latrunculin A to actin, and K_dLP for profilin to the complex of actin and latrunculin A, and also to k_A, k_AP, k_AL, and k_ALP, the rate constants of ATP dissociation from actin, profilin, actin-latrunculin A, and actin-latrunculin A-profilin ternary complex, respectively.

Native Gel Electrophoresis-- Actin at a concentration of 2.9 µM was incubated in Buffer G with or without 3.4 µM thymosin ₄ in the presence and absence of 40 µM latrunculin A. Solutions were incubated for 35 min before loading on gel. Native gels were equilibrated in buffer containing 0.1 mM CaCl₂, 0.01% sodium azide, 0.2 mM ATP, 0.2 mM dithiothreitol, and 25 mM Tris-Tricine, pH 8.2. In experiments with labeled thymosin ₄, the picture of the fluorescent gel was taken before staining with Coomassie.

Fluorescence Anisotropy-- Data were collected on a Photon Technology International (South Brunswick, NJ) spectrofluorometer. Tetramethylrhodamine-5-maleimide-labeled thymosin ₄ was excited with vertically polarized light at 546 nm. The horizontal and vertical components of the emitted light were detected at 568 nm. Solutions of labeled thymosin ₄ (0.1 µM) in Buffer G were titrated with Mg²⁺-actin in the presence or absence of a constant amount of latrunculin A (or with latrunculin A in the presence or absence of a constant amount of Mg²⁺-actin).

Data were fit globally as described by Vinson et al. (19), with the inclusion of a term for the formation of a ternary complex between actin, latrunculin, and thymosin. Fitting parameters included the equilibrium dissociation constants for thymosin ₄ to actin (K_dT), for latrunculin A to actin (K_dL), and for thymosin ₄ to the complex of actin and latrunculin A (K_dLT) and the terms indicating the anisotropy of free thymosin ₄ (r_f) and the anisotropy of the complex of thymosin ₄ with actin or with actin-latrunculin A complex (r_b). Assuming that the concentration of free thymosin, [T], is low relative to K_dT, (or strictly, [T]/K_dT (1+ [L]/K_L), equations for the observed fluorescence anisotropy, r, can be written as a function of the total actin, [A]_t, and total latrunculin A, [L]_t, concentrations as follows,

(Eq. 1)

(Eq. 2)

where [A] is free actin concentration.

Analytical Ultracentrifugation-- Sedimentation equilibrium experiments were performed using absorption optics with data collected at 535 nm (the absorption maximum for labeled thymosin ₄) in a Beckman XLA centrifuge. All samples contained 1.6 µM labeled thymosin ₄. Samples of 110 µl in Buffer G reached equilibrium in 42 h at 13,900 rpm (after initially overspeeding to 15,100) at 4 °C. Buffer density was determined by pyknometry, and partial specific volumes were as previously reported for actin or calculated from amino acid sequence for thymosin ₄ (20). The gradient was analyzed according to a method of implicit constraints as described previously (21). In brief, at 535 nm, only labeled thymosin ₄ has a measurable extinction coefficient. The other sample components are invisible. Therefore, at this wavelength, the absorbance at any radius is directly proportional to the sum of the concentration of all allowable thymosin ₄-containing species (in a model of noncompetitive inhibition, these include thymosin ₄, thymosin ₄ bound to actin, and thymosin ₄-actin-latrunculin A ternary complex). The species are assumed to be in chemical equilibria at all radii as governed by appropriate equilibrium dissociation constants. Curve fitting is constrained by the initial concentration of all components, and the fitting parameters include only the dissociation constants and the concentration of each component at an arbitrary radius, r_b (21).

Measurement of DNase I Activity-- DNase I (30 nM) was incubated with actin (30 nM) with varying concentrations of latrunculin A for 5 min at room temperature before adding 100 µg/ml DNA. The reaction mixture was in buffer containing 21 mM NaCl, 0.1 mM CaCl ₂, 2.0 mM MgCl₂, 0.1 mM ATP, and 5 mM Tris, pH 7.9. After 20 min, samples were loaded on 0.7% agarose gels, and the gels were subsequently stained with ethidium bromide.

Crystallization of Latrunculin A and Actin-- Crystals were grown in hanging droplets containing 1.3-1.5 M ammonium sulfate, 3 mM MgCl₂, 60 mM imidazole, pH 6.7, with actin concentration at 9 mg/ml and a ratio of 1:1 or 1.2:1 of latrunculin A to actin. Crystals appeared after 2-4 weeks at room temperature. Seemingly identical crystallization conditions produced satisfactory crystals only in approximately 50% of attempts. Crystals with typical dimensions 0.4 × 0.5 × 0.4 mm were wet mounted on glass capillaries at room temperature. The data were collected using Cu K radiation ( = 1.541Å) from a Rigaku RU-200 x-ray generator (40 kV, 90 mA, 0.3 mm collimator). The generator was equipped with an R-Axis IIc image plate, and data were collected at a crystal-IP distance of 100 mm with an oscillation range of 1.1 degree/frame. Data were integrated and scaled using the two companion programs of the HKL Suite, Denzo and Scalepack (22).

Crystal Density Measurements-- Stock 50 or 60% Ficoll solutions in crystallization buffer, prepared according to Ref. 23, were mixed in the desired ratios with crystallization buffer to prepare solutions of various Ficoll concentrations. The density of each solution was calculated from the mass of 0.4 ml of solution as measured with a positive-displacement pipette. Ammonium sulfate was used to vary the buffer density for a given concentration of Ficoll. The technique relies on the assumption that ammonium sulfate, but not Ficoll, rapidly enters the solute component of the crystal. Crystals were layered on top of a step gradient of Ficoll in a 6-mm-diameter glass cuvette and immediately centrifuged at 8000 × g for 2 min. After the positions of crystals were located, the cuvette was centrifuged for an additional 1 min to check for changes in position. In control experiments, centrifugation time varied from 1 to 10 min. Some crystals were lightly cross-linked in 0.15% glutaraldehyde for 12 h at room temperature prior to density measurements. Whereas uncross-linked crystals were stable for only 10-15 min at low solvent density, the crystals were stable (with constant density) for several hours after covalent cross-linking. Crystal density as a function of the partial specific volume of the protomer was then calculated as described previously (23).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Steady State Fluorescence Data for F-actin Are Consistent with Formation of a 1:1 Complex of Latrunculin A and Actin with Little or No Dependence on Ionic Strength-- The calculated concentration of latrunculin A-actin complex was proportional to the concentration of latrunculin A, consistent with monomer sequestration (Fig. 1, inset). The calculated equilibrium dissociation constant (K_dL) is similar to that previously reported (K_dL = 0.2 µM in very low ionic strength buffer containing 0.1 mM CaCl₂ and 2.0 mM MgCl₂) (2). Latrunculin A did not cause any significant differences in the slopes of the curves for fluorescence versus actin concentration relative to controls, consistent with 1) the absence of actin-filament capping activity (24), and 2) similar binding affinity to pyrenyl-actin and unlabeled actin (25). The slight increase (or perhaps absence of change) in affinity at increasing ionic strength implies that electrostatic interactions contribute insignificantly to binding (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Steady state determination of apparent critical concentration and equilibrium dissociation constant for latrunculin A as a function of salt concentration. The KdL is calculated indirectly from the change in apparent critical concentration of 4% pyrenyl-Mg2+-actin caused by 0.25 µM latrunculin A. Error bars represent ± 2 S.D. for measurements from three different actin preparations. The line through the data is arbitrary. Inset, steady state fluorescence data after 24 h in 10 mM KCl for 0 (filled circles), 0.25 (circles), 0.5 (triangles), and 0.75 µM (squares) latrunculin A. The lines through the data represent a least squares best fit. Error bars represent ± 2 S.D. for three samples made from the same F-actin stock.

The Initial Rate of Polymerization in a Seeded Polymerization Assay Fit a Model in Which the Latrunculin A-Actin Complex Did Not Participate in Elongation-- Unlike the actin-monomer-binding protein, profilin, elongation data for actin in the presence of latrunculin A can be explained by monomer sequestration alone with K_dL of 0.22 ± 0.06 µM (Fig. 2). Notably, although the data suggest that monomer sequestration is the most simple explanation for these data, they fail to prove that latrunculin A-actin complex does not participate in elongation, as any of a number of more complicated models are plausible in which the complex adds and dissociates in a nonproductive manner.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Dependence of initial rates of seeded actin polymerization on latrunculin A concentration. Total actin concentration was 4 µM. The squares, circles, upward-pointing triangles, and downward-pointing triangles represent four independent sets of data. The line represents the best simultaneous fit to all four sets of data assuming a critical concentration of 0.08 µM and a model of monomer sequestration by latrunculin A, in which case, KdL is 0.22 µM.

Profilin and Latrunculin A Bind Noncompetitively to Actin-- Nucleotide exchange on actin was used to indirectly assess the interaction of latrunculin A with actin in the presence or absence of profilin (profilin, by itself, accelerates nucleotide exchange on actin (18)). Latrunculin A alone inhibited nucleotide exchange on 1.7 µM Mg²⁺-actin, as previously reported (Fig. 3, inset) (3). Latrunculin A also inhibited nucleotide exchange in the presence of profilin (Fig. 3), implying either that latrunculin A bound competitively with profilin to actin or that nucleotide exchange on actin was inhibited in the ternary complex of latrunculin A, profilin, and actin. Quantitative evaluation of the data eliminated the possibility that binding was competitive. Consistent with fluorescence anisotropy data (data not shown), profilin binds to actin with equilibrium dissociation constant, K_dP, of 0.1 µM under these experimental conditions. Assuming this K_dP and a model of competitive binding, the data could not be fit by any possible combination of binding constants of latrunculin A to actin and exchange rates for the complexes of profilin-actin and latrunculin A-actin (Fig. 3, dashed line). Also, the best possible fit required an unreasonable K_dL for latrunculin A-actin (0.009 µM) when compared with the other results reported here. In contrast, a model in which profilin and latrunculin A bound independently to actin provided a good fit to the experimental data and yielded a reasonable K_dL for latrunculin A-actin (0.28 µM; in Table I, the large error estimate for K_dL in the nucleotide exchange experiment relative to the other experimental methods reported here is due to the large number of fitting parameters). Moreover, the best possible global fit to the data was achieved when latrunculin A and profilin were allowed to interact cooperatively with actin, so that the affinity of latrunculin A for actin was increased by a factor of 1.8 when profilin was bound. The fit achieved by allowing this minor degree of positive cooperativity (Hill coefficient of 1.1) was not significantly improved in comparison to a more simple, independent binding model. We conclude that the data rule out competitive binding, but the presence of either slight positive cooperativity or no cooperativity can plausibly explain the experimental results.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effects of profilin and latrunculin A on the rate of nucleotide exchange on actin. Rate of nucleotide exchange in the presence (triangles) and absence (circles) of 0.2 µM profilin is shown as a function of latrunculin A concentration. The inset shows the bottom curve with a magnified scale. The data are fit assuming that profilin and latrunculin A bind independently to actin (solid lines) or competitively to actin (dashed lines). Error bars represent ± S.E. for three samples.

View this table: [in this window] [in a new window]   Table I Equilibrium dissociation constants organized by experimental technique Equilibrium dissociation constants are listed for binding latrunculin A to actin (KdL), thymosin 4 to actin (KdT), thymosin 4 to actin saturated with latrunculin A (KdLT), and profilin to actin (KdP). Data used for calculation of each constant are shown in the figures indicated. Error estimates are based on a standard least squares deviation fitting algorithm with 95% confidence limits.

For the nucleotide exchange experiments, profilin concentration was constant (0.2 µM) and not saturating; therefore, the exchange rate with no latrunculin A (0.023 s¹) is not equal to k_AP; rather, k_AP was obtained as the best global fit to the data. Assuming noncompetitive binding, the best global fit for the exchange rate constants was obtained with k_AP = 0.137 s¹, k_A = 0.0015 s¹, k_AL = 0.0003 s¹, and k_ALP = 0.0011 s¹. Values for k_A and k_AP are consistent with previous reports (26). The fit curves are insensitive to relatively large changes in k_AL and k_ALP, and these parameters cannot be distinguished from 0 by the given data.

Native Gel Electrophoresis Provides Qualitative Evidence That Latrunculin A Inhibits Binding of Thymosin ₄ to Actin-- The addition of latrunculin A to samples containing mixtures of thymosin ₄ and actin caused less actin to shift to a band corresponding to a high electrophoretic mobility complex of thymosin ₄ and actin, implying that the complex is dissociated by latrunculin A (Fig. 4, compare lanes 3 and 4). Similarly, less fluorescently labeled thymosin ₄ shifted to the band corresponding to this complex in the presence of latrunculin A (Fig. 4, compare lanes 1 and 2). Previous results have suggested that the extent of binding seen in this assay may not quantitatively reflect the apparent K_d for thymosin ₄ and actin (27), perhaps because of excluded volume effects, but changes in the amount of shifted protein are qualitatively indicative of the extent of formation of a thymosin ₄-actin complex. The data also show that unlabeled thymosin ₄ bound as well to actin as labeled thymosin ₄ and that binding to actin was inhibited by latrunculin A to the same extent as covalently labeled thymosin ₄ (Fig. 4, lanes 7-10).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Qualitative assessment of competition between thymosin 4 and latrunculin A for actin using a native gel. Actin (2.9 µM) was incubated with (lanes 1-4, 7, and 8) or without (lanes 5, 6, 9, and 10) thymosin 4 (3.4 µM) and run on a nondenaturing electrophoretic gel. In lane 1, fluorescently labeled thymosin 4 separates into two bands. The low mobility band at the top is free thymosin 4, and the high mobility band is thymosin 4 that is bound to actin. In lane 2, 40 µM latrunculin A is also present, and much less thymosin 4 is bound to actin. The identical gel samples are shown in lanes 3 and 4 after Coomassie staining, in which actin is the primary protein visualized. In lane 3, actin is shifted to the same position as actin-bound thymosin 4. Latrunculin A causes less actin to shift (lane 4). Samples of actin alone and actin with latrunculin A are shown as unshifted controls in lanes 5 and 6, respectively. The samples in lanes 7-10 are identical to those in lanes 3-6 except that the thymosin 4 is unlabeled.

Experiments Providing Quantitative Data Show That Latrunculin A Decreases the Affinity of Thymosin ₄ for Actin by Approximately 1 Order of Magnitude-- Fluorescence anisotropy of labeled thymosin ₄ increased from 0.08 when free to 0.18 when saturated with actin. The anisotropy was lower in the presence of latrunculin A than in its absence at any given actin concentration, indicating that latrunculin A inhibits binding of thymosin ₄ to actin (Fig. 5A, top panel). Increasing latrunculin A at fixed actin concentration caused dissociation of thymosin ₄ from actin (Fig. 5A, bottom panel); if binding was independent (that is, if K_dT is equal to the equilibrium dissociation constant for binding of thymosin ₄ to latrunculin A-actin complex, K_dLT), then these curves would be flat. In contrast, the best global fit to all four data sets was obtained with K_dT = 0.23 ± 0.02 µM, K_dL = 0.35 ± 0.05 µM, and K_dLT = 7.65 ± 0.74 µM. Therefore, this assay implies inhibition by latrunculin A, with approximately 33 times (the ratio of K_dLT to K_dT) weaker affinity of thymosin ₄ for actin when latrunculin A is bound to actin.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Quantitative assays of competition between thymosin 4 and latrunculin A for actin. A, fluorescence anisotropy showing titration of 0.1 µM solutions of labeled thymosin 4 with actin in presence (triangles) and absence (circles) of 30 µM latrunculin A (top panel) and titration of 0.1 µM solutions of labeled thymosin 4 with latrunculin A in presence of 1 µM (squares) and 5 µM (triangles) actin (bottom panel). The solid lines show the result of a global fit to the data, with KdT = 0.23 µM, KdL = 0.35 µM, and KdLT = 7.65 µM. B, the initial rate of seeded actin polymerization is shown as a function of the concentration of latrunculin A (open squares), thymosin 4 (filled circles), or in the presence of a fixed concentration of latrunculin A (2.0 µM) as a function of the concentration of thymosin 4 (filled triangles). Total actin concentration was 4 µM. Solid lines represent the best simultaneous fit to all three sets of data assuming competitive binding, with KdL = 0.40 µM and KdT = 0.31 µM. The dashed line shows the best fit assuming independent binding, with KdL = 0.30 µM and KdT = 0.26 µM. The same data obtained at fixed latrunculin A concentration and various concentrations of thymosin 4 are replotted as a function of the sum of the concentration of latrunculin A and thymosin 4 (open triangles) to illustrate that the mixture of the two ligands yields essentially indistinguishable results from those obtained if either component was replaced with the other. This would be predicted if thymosin 4 and latrunculin A bind to actin with similar Kd and if binding is competitive, but not if binding was independent. C, sedimentation equilibrium gradients for 1.6 µM labeled thymosin 4 (triangles), 1.6 µM labeled thymosin 4 and 3.0 µM actin (circles), and 1.6 µM labeled thymosin 4, 3.0 µM actin, and 8.0 µM latrunculin A (squares). Only the labeled thymosin 4 is visible at the recorded wavelength, and the absorbance at any radius is therefore directly proportional to the concentration of thymosin 4. The different gradients reflect different apparent molecular weights of thymosin 4 resulting from complex formation with the other ligands. The shallower gradient observed after addition of latrunculin A reflects inhibition of binding between actin and thymosin 4, resulting in less actin bound to thymosin 4 and therefore a lower average apparent molecular weight. The solid lines show the result of a global fit to the data assuming molecular weights consistent with atomic formulae and a noncompetitive model of inhibition, with KdT = 0.92 µM, KdL = 0.52 µM, and KdLT = 8.0 µM. The differences between the actual data and the theoretical fit are shown in the three top panels.

Measurement of the elongation rate after seeding actin polymerization provides additional information regarding the interaction of latrunculin A, thymosin ₄, and actin (Fig. 5B). The data obtained for equivalent amounts of latrunculin A and thymosin ₄ were nearly indistinguishable, therefore implying that the effects of thymosin ₄ on filament elongation, like those of latrunculin A, can be explained by a simple model of monomer sequestration and that the binding constants K_dL and K_dT are similar. The observation that latrunculin A and thymosin ₄ have an additive effect on actin polymerization rates is demonstrated graphically in Fig. 5B, consistent with competitive binding by the two ligands on actin. If binding of the ligands occurred independently, then the effect of a mixture of components would be less than the effect of either component alone at a concentration equal to the sum of the components. Quantitative analysis of the elongation data confirms that a competitive binding model, but not an independent binding model, adequately explains all the data for both ligands. The best global fit to the data, assuming competitive binding with a critical concentration of 0.08 µM, yields K_dL = 0.40 ± 0.05 µM, K_dT = 0.31 ± 0.03 µM. Although these data do not require a more complicated model that includes a finite K_dLT to generate a good fit, the results are insufficiently sensitive to distinguish between noncompetitive inhibition (finite K_dLT) and a more simple competitive binding model (infinite K_dLT).

Sedimentation equilibrium experiments also graphically illustrate inhibition of the thymosin ₄ ligand by latrunculin A (Fig. 5C). Samples were made with 1.6 µM labeled thymosin ₄ with or without 3.0 µM actin and either 8.0 µM latrunculin A or an equivalent volume of Me₂SO. Use of labeled thymosin ₄ allows for large and easily detectable changes in the apparent molecular weight of thymosin ₄ upon binding to the much larger molecule, actin. The observation of a steeper exponential gradient in a sample of thymosin ₄ with actin than for thymosin ₄ alone is therefore due to the formation of an actin-thymosin ₄ complex (Fig. 5C, compare circles and triangles). If binding of the thymosin ₄ and latrunculin A ligands on actin occurs independently, then the data with or without latrunculin A would be indistinguishable (Fig. 5C, compare circles and squares). Rather, binding of thymosin ₄ to actin was inhibited by latrunculin A, with the following best estimates for equilibrium dissociation constants: K_dL = 0.52 ± 0.18 µM, K_dT = 0.92 ± 0.28 µM, and K_dLT = 8.0 ± 1.9 µM. The data for thymosin ₄ alone demonstrate a slight extent of systematic deviation from the theoretical curve, consistent with a small amount of dimeric protein (3% dimer according to an analysis not shown). Although native thymosin ₄ has been reported to be monomeric (28), this small extent of dimerization may have previously escaped detection or be a unique result related to bacterial expression of protein or covalent labeling. Perhaps dimerization of thymosin ₄ explains the systematic deviation observed in the plots for the data that includes actin, with a small amount of dimeric thymosin ₄ binding to two actin molecules (only visible at large radius and high actin concentrations).

The results of all quantitative assays for latrunculin A-actin interactions are shown in Table I. The variation of reported equilibrium dissociation constants in Table I probably reflects limitations of the techniques and true differences related to differences between binding to Ca²⁺-actin and Mg²⁺-actin and difference in ionic strength, but it is also possible that they reflect true pressure-related changes that occur during analytical centrifugation.

Latrunculin A Did Not Inhibit the Binding of DNase I to Actin-- The endonuclease activity of DNase I is inhibited by actin. Measurement of the activity of DNase I by assay of DNA fragmentation has previously been used to show that the actin-binding protein gelsolin can displace actin from DNase I (29). Using the same assay, we were unable to detect any increase in the activity of DNase I in the presence of saturating concentrations (up to 20 µM) of latrunculin A (data not shown). A control using DNase I with or without latrunculin A ruled out the possibility that latrunculin A was by itself an inhibitor of DNase I. The high affinity interaction between actin and DNase I (K_a of approximately 10¹⁰ M¹ (29)) limits the detection of small changes in affinity between DNase I and actin. A 6-fold drop in affinity with the given experimental conditions (30 nM DNase I and 30 nM actin) would be predicted to increase the amount of free DNase I from 1.7 to 4.0 nM. Control experiments determined that the assay conditions could reliably detect a change of this magnitude. Therefore, we conclude that latrunculin A does not inhibit DNase I-actin interactions, or if it does so, the inhibition is minimal.

Inhibition of Polymerization by Latrunculin A Results in the Formation of a Unique Actin Crystal-- Crystals were shown to contain both latrunculin A and actin by mass spectroscopy and gel electrophoresis, respectively. X-ray diffraction resulted in a data set that was 99.9% complete for the range 40.0-3.2 Å with an overall R_merge (_hi·I_h, i · <I_h> ·/_{h i} I_{h, i}) of 11.2%, and for the highest resolution shell, 3.44-3.34 Å, the R_merge was 32.2%, with an average I/I = 3.5 (Fig. 6, A and B). The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 101.68, b = 103.09, and c = 127.12 Å;  =  =  = 90.0°. Systematic absences were consistent with the space group assignment of P2₁2₁2₁ (for h00, h  2n; for 0k0, k  2n; and for 00l, l  2n). At least 12 systematically absent reflections were measured for each of the three axes. Detailed summaries of the data collection statistics are shown in Table II. The crystals diffracted to 3.10 Å or better at 300 K; however, a complete data set could be processed only up to 3.2 Å using two crystals. All crystals examined (n = 5), however, resulted in data consistent with orthorhombic space group P2₁2₁2₁ and similar unit cell dimensions. Crystal density was consistent with eight molecular complexes per unit cell (Fig. 6C). Calculation of the Matthews coefficient for a crystal with assumed actin:latrunculin A stoichiometry of 1:1 resulted in the V_M value of 3.94 Å³/dalton with 72% solvent content if two protomers are assumed to be in the asymmetric unit (30). These values are within the range observed for other proteins. We are currently calculating the self-rotation function to ascertain the observed symmetry and to look for any other noncrystallographic symmetry elements.

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Crystals of actin and latrunculin A. A, photograph of actin-latrunculin A crystal measuring 0.75 mm in its longest dimension. B, diffraction pattern obtained from pictured crystal. The highest resolution seen is approximately 3.10 Å (inset). C, results of crystal density measurements in step gradients of Ficoll using either intact crystals (solid triangles) or crystals cross-linked with glutaraldehyde (open squares). Each data point represents the result for a single crystal, and the error bars define the upper and lower limits of the step in the gradient at which the crystal reached equilibrium. Variation along the x axis is achieved by varying the sample concentration of ammonium sulfate. The best fit with n set to 8 (or two protomers per asymmetric unit) gives a partial specific volume of  = 0.765 ml/g (solid line).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our current data show that in the presence of latrunculin A, profilin binds to actin independently, or perhaps with some positive cooperativity. Similarly, latrunculin A had no apparent effect on actin-DNase I interactions. In contrast, binding of thymosin ₄ to actin is inhibited by latrunculin A. Previous reported information suggested that latrunculin A bound to actin in the cleft between subdomains 2 and 4 of actin, at a site adjacent to the adenine nucleotide binding site (3). We confirm that latrunculin A inhibits adenine nucleotide exchange on actin. Just as DNase I, which bridges the cleft between subdomains 2 and 4, reduces the rate of adenine nucleotide exchange on actin (31), latrunculin A may limit the flexibility of the cleft and trap nucleotide, thereby resulting in relative inhibition of nucleotide exchange.

Inhibition of the binding of thymosin ₄ to actin by latrunculin A does not necessarily conflict with the postulated localization of latrunculin A to the nucleotide-binding cleft of actin. Neither the analytical ultracentrifuge data nor the fluorescence anisotropy data are consistent with simple competitive inhibition (in which case, (K_dLT)¹ = 0), but rather, they suggest that latrunculin A inhibits thymosin ₄ noncompetitively. Noncompetitive inhibition of thymosin ₄ binding to actin by latrunculin A has several possible structural interpretations. Latrunculin A could bind near or at the binding site of thymosin ₄, but not be large enough to create a steric effect that completely eliminates simultaneous binding by thymosin ₄. Because evidence has been reported that thymosin ₄ may bind to actin in an extended conformation, with interactions on actin at multiple sites (5), a second possibility is that latrunculin A sterically competes at one site of interaction, and the weak affinity of thymosin ₄ in the presence of latrunculin A is due to residual interactions between actin and thymosin ₄ at other sites. The third possibility is that latrunculin A inhibits binding by an allosteric effect, perhaps with a binding site in the nucleotide binding cleft of actin, as previously suggested.

The most recent models of thymosin ₄ bound to actin show that thymosin ₄ is more than 20 Å from the nucleotide binding cleft (5, 8). In these analyses, earlier evidence that thymosin ₄ could be covalently cross-linked to ATPS (32) was considered to be an artifact produced by cross-linking free, rather than actin-bound, nucleotide to thymosin ₄. Although considerable evidence supports the identification of an interaction between subdomain 1 of actin and thymosin ₄ (5, 32, 33), disagreement continues regarding whether thymosin ₄ binds directly to subdomain 2 of actin (5) or whether the effects on subdomain 2 can be explained by an allosteric mechanism (33). To summarize, if latrunculin A does bind in the nucleotide-binding cleft of actin, then effects on thymosin ₄ are the result of substantial allosteric effects on actin. Conversely, latrunculin A may bind at or near one of the thymosin ₄ binding sites on actin and, like thymosin ₄, allosterically affect the rate of nucleotide exchange.

Proteins that bind to actin may regulate actin filament dynamics and may have functions independent of their actin-regulatory functions. Moreover, these functions may be regulated by their interaction with actin filaments or monomers. Profilin and thymosin ₄ have proven to be typical examples for which identification of actin-regulatory functions has been followed by the identification of complex functions that may, in turn, be dependent on actin binding activity. The regulation of phosphoinositide metabolism by profilin and the roles of thymosin ₄ in wound healing, apoptosis, and immunosuppression illustrate the diversity of functions served by such proteins (12, 13, 11, 34). With latrunculin A, we have demonstrated that a drug that binds to actin may have differential effects on various actin-binding proteins. Given the independent functions of these actin-binding proteins, the effects of actin-binding drugs may have far-reaching consequences. First, these effects may be significant when the drugs are employed to dissect cell biological problems. Thus, the consequences of addition of latrunculin A to cells in vivo or in situ may be the result of factors other than simple monomer sequestration. Secondly, our preliminary work with more than 20 derivatives of latrunculin A suggests that derivatization may independently alter properties of monomer sequestration and inhibition of thymosin ₄ binding. Finally, it may be possible to exploit these effects in a therapeutic context.

With regard to the effects of thymosin ₄ on immune suppression, thymosin ₄ sulfoxide appears to be an effector molecule for the anti-inflammatory effect of glucocorticoids (13). It is not known how thymosin ₄ gets sulfonated or gets to the extracellular space, but it is certainly possible that only free thymosin ₄ actively participates in either one or both of these steps. If so, latrunculin A may activate the anti-inflammatory function of thymosin ₄ by increasing free thymosin ₄ concentrations. Thus, actin-binding drugs may induce specific, desired effects by a novel mechanism. Because several cell regulatory proteins, such as protein kinases and their substrates, are spatially regulated by anchorage to F-actin (35-37), the potential displacement and activation of these proteins by actin-binding drugs suggests that this general mechanism may, in other instances, have broad implications with complicated consequences.

Latrunculin A lowers the affinity of actin for thymosin ₄ by approximately 1 order of magnitude. Although this is not a large thermodynamic effect, it is likely to be significant in living cells. In a cell such as a polymorphonuclear leukocyte with approximately 150 µM thymosin ₄ (38), the addition of saturating concentrations of latrunculin A would result in a rapid change in the effective equilibrium dissociation constant for thymosin ₄ from approximately 0.3 (K_dT) to 8 µM (K_dLT). Assuming that the critical concentration of actin in a cell is maintained at approximately 1.0 µM by capping of barbed ends, this would be expected to cause an immediate increase in the amount of free thymosin ₄ by approximately 15 µM. If excluded volume conditions in the cytoplasm increase the relative affinity of these interactions by a factor of 10, which is a conservative estimate (39), then the concentration of free thymosin ₄ would increase 3-fold immediately after adding latrunculin A. Later, as actin depolymerizes and the complex of actin-latrunculin A begins to accumulate, this complex becomes a sink for monomer sequestering proteins, particularly those that bind to the complex as well as they bind to free actin. Thus, at steady state, both free profilin and free thymosin ₄ concentrations would be diminished after addition of latrunculin A, but because of the differential effect of latrunculin A, the decrease in profilin concentration would be much greater than that for thymosin ₄.

The actin crystal described here is unique because it is the first actin crystal to diffract to this resolution in the absence of other actin-binding proteins. It is also unique in its symmetry and packing volume. The successful use of a marine natural product inhibitor of actin polymerization to stabilize actin for purposes of crystallization may have other applications. In cases in which production of an actin crystal in complex with other proteins or peptides of interest has been unsuccessful to date, the addition of latrunculin A or another actin-stabilizing marine natural product may facilitate crystal growth.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the Medical Research Service of the Department of Veteran Affairs. To whom correspondence should be addressed: Box 100277, Dept. of Medicine, University of Florida, Gainesville, FL 32610. Tel.: 352-392-4059; Fax: 352-392-6481; E-mail: bubb@medicine.ufl.edu.

Published, JBC Papers in Press, June 19, 2000, DOI 10.1074/jbc.M004253200

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