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Originally published In Press as doi:10.1074/jbc.M002939200 on May 5, 2000

J. Biol. Chem., Vol. 275, Issue 29, 22470-22478, July 21, 2000
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An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin*

Vicci L. KormanDagger , Victoria Hatch§, Kavara Y. Dixon, Roger Craig||, William Lehman§, and Larry S. TobacmanDagger **

From the Departments of Dagger  Biochemistry and  Internal Medicine, University of Iowa, College of Medicine, Iowa City, Iowa 52242, § Department of Physiology, Boston University School of Medicine, and the || Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Received for publication, April 6, 2000, and in revised form, May 4, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca2+ to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca2+ showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiac and skeletal muscle contraction is controlled by a complex allosteric system in which myosin-actin interactions are regulated by tropomyosin and troponin. Tropomyosin is an extended, dimeric coiled-coil and spans seven actin monomers on the thin filament. Troponin is composed of three subunits: troponin T connects the other two subunits and tropomyosin to the actin filament, troponin I inhibits myosin binding to actin, and troponin C serves as a Ca2+ sensor for the system (for reviews see Refs. 1 and 2). Structural studies have shown that tropomyosin can bind to three different regions of the actin filament (3-7). In the absence of Ca2+, tropomyosin appears to bind to subdomain 1 of actin and to bridge over subdomain 2 to the next actin monomer (4). In the presence of Ca2+, tropomyosin moves toward subdomains 3 and 4 (3) and moves even further in the presence of myosin (8). Whereas these and other results support a three state model of muscle regulation involving tropomyosin movement (9-12), information defining the position and interactions of troponin on the filament and actin residues that specifically interact with the regulatory proteins is limited.

One strategy to examine muscle protein-protein interactions has been to exploit functionally altered mutants (13-15). For example, a Drosophila melanogaster actin subdomain 2 mutant, E93K, which inhibits tropomyosin-actin sliding in isolated protein preparations, has been utilized to investigate tropomyosin-based regulation (16). These and other data (17-20) suggested that actin subdomain 2 may be important for tropomyosin and troponin function. Recently, we have shown that Saccharomyces cerevisiae actin can serve as a model system for examining troponin-tropomyosin-mediated regulation (21). This system is advantageous, because actin mutants are available in greater quantity than from Drosophila. A number of actin surface residue mutations can be used to examine sites that interact with the regulatory proteins to influence thin filament conformation. In the present study, yeast actin subdomain 2 mutant D56A/E57A (22) was used to assess the interactions of these residues with tropomyosin and troponin. Substitution of alanine for charged amino acids at these positions increased tropomyosin affinity for actin 3-fold, supporting the view that ionic forces influence tropomyosin-actin interaction (6, 23-25). Regulated thin filaments containing troponin, tropomyosin, and D56A/E57A actin did not exhibit Ca2+-mediated activation of myosin S1 ATPase rates and had decreased affinity for myosin S1. Electron microscopy and three-dimensional image reconstruction indicated that, despite these inhibitory properties of regulated mutant actin filaments, the Ca2+-induced shift in tropomyosin position on actin was normal and not different from that found for regulated muscle actin filaments. The results suggest that charged residues on subdomain 2 of actin are important for myosin binding, regulatory protein binding, and thin filament regulation. They also suggest the importance of the strength of myosin binding for thin filament activation.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Protein Purification-- Bovine cardiac troponin and tropomyosin were extracted from ether powder using the procedure of Tobacman and Lee (26). Myosin S1 was purified from rabbit fast skeletal muscle following the method described by Weeds and Taylor (27). Actin was purified from rabbit fast skeletal muscle as reported by Spudich and Watt (28). The D56A/E57A actin strain, a gift from Dr. Drubin's laboratory (22), was grown to stationary phase at 25 °C. The wild type actin was purified from commercial bakery cakes of Saccharomyces carlsbergensis, which has an actin sequence identical to that in S. cerevisiae (29). Both actins were purified using DNase columns based on the method by Cook et al. (29).

Ca2+ G-actin was converted to Mg2+ G-actin using the method of Strelezka-Golaszewska et al. (30). The Ca2+ or Mg2+ G-actin was then polymerized by adding 3 mM MgCl2. In the case of the Ca2+ G-actin, polymerization by Mg2+ addition causes a heterogeneous population of Ca2+ or Mg2+ F-actin, here termed conventional F-actin. Similar to other actins in which the amino acid residues of subdomain 2 have been modified (17, 19) or mutated (16), polymerization of D56A/E57A actin was altered under some conditions. Polymerization was normal in the presence of 3 mM MgCl2 and low ionic strength, but it was necessary to add phalloidin to prevent depolymerization of the mutant filaments under the high salt conditions of the co-sedimentation binding assays (data not shown). Wild type actin was treated similarly.

Co-sedimentation Actin-binding Assay-- Bovine cardiac tropomyosin was labeled with [H3]iodoacetate at residues Cys190 (24). The assay conditions are described in the figure legends. Samples were centrifuged at 35,000 rpm using a TLA100 rotor (Beckman) for 30 min at 25 °C after a 30-min incubation. Aliquots were taken before and after centrifugation to compare the total radioactivity in the starting reaction mixture with the remaining radioactivity in the supernatant. A linear lattice equation was used to analyze the data (31, 32). In this equation, Ko symbolizes the affinity of one extended ligand (tropomyosin) for an isolated binding site on a linear lattice (actin), and y is a measure of cooperativity. Specifically, y is the ratio describing the probability for tropomyosin to bind adjacent to another bound tropomyosin, relative to the probability that it binds in an isolated position (32). Kapp is approximately equal to the product of Ko and y.

Myosin S1 ATPase Assay-- Myosin S1 ATPase activity was determined using the procedure of Pollard and Korn (33). The following conditions were used: 25 °C, 7 µM phalloidin-stabilized yeast F-actin, 1 µM rabbit fast skeletal muscle myosin S1, either 2 µM bovine cardiac tropomyosin or 1.2 µM tropomyosin plus 1.2 µM bovine cardiac troponin, 5 mM imidazole (pH 7.5), 3.5 mM MgCl2, 7.5 mM KCl, 1 mM dithiothreitol, and either 0.5 mM EGTA or 0.2 mM CaCl2. A total of six data points were taken at 2-min intervals. The data were corrected for myosin S1 ATPase activity in the absence of actin.

Myosin S1 Binding Assays-- Strong myosin binding decreases the fluorescence intensity of actin that is labeled by pyrene at Cys374 (34). The affinity (K', below) of myosin S1-ADP for pyrene-labeled muscle filaments was found by serial additions of myosin S1, with the data fit to a simple hyperbolic binding model. Under the tight binding conditions of the experiments, hyperbolic binding also approximated the behavior of regulated pyrene actin filaments (see Fig. 3A and Table II). Data in the presence of very low S1 concentrations presumably show cooperativity but is not relevant to the competition experiments.

Modest yields of the yeast actins made it difficult to label sufficient quantities with pyrene for direct measurement of myosin S1 thin filament binding. Instead, a competition assay was used to measure myosin S1-ADP affinity for filaments containing wild type or mutant yeast actin. 0.8 µM myosin S1 was added to pyrene-labeled muscle actin or regulated actin filaments, and the fluorescence change was noted. Then, serial additions of an unlabeled competitor actin or thin filament were made to displace the myosin S1 from the labeled filaments. Data from duplicate titrations were corrected for dilution, averaged, and normalized. Saturating concentrations of myosin S1 quenched the fluorescence by 86%.

The data were analyzed according to a simple competition model. Binding to the labeled actin is described by K' = A'S/(A' × S), and binding to the unlabeled actin is given by K = AS/(A × S). A is the free unlabeled actin, A' is the free labeled actin, S is the free myosin S1, AS is the unlabeled actin-myosin S1 complex, and A'S is the labeled actin-myosin S1 complex. A'S is the physically relevant root of the following cubic expression in A'S, derived from conservation of mass relationships and the above expressions for K and K',
0=[(Q×(S<SUB>T</SUB>−A′S)−A′S)×(Q+K×A′S)]−Q×K×A<SUB>T</SUB>×A′S,

Q≡[(K′×A′<SUB>T</SUB>)−(K′×A′S)] (Eq. 1)
where ST is the total myosin S1, and AT and A'T are the total unlabeled and labeled actins, respectively. Using Equation 1 for A'S, the curvefitting program SCIENTIST was used to determine the value of K resulting in the best fit of the fluorescence data to the following equation,
F=F<SUB>e</SUB>−(A′S/A′<SUB>T</SUB>)×(F<SUB>e</SUB>−F<SUB>s</SUB>) (Eq. 2)
where Fe is the fluorescence of the empty actin filament (i.e. with no myosin bound), and Fs is the fluorescence of the actin filament with saturating myosin S1.

The conditions of the myosin binding experiments were: 25 °C, 1 µM pyrene-labeled rabbit fast skeletal muscle actin (34), 20 mM imidazole (pH 7.5), 0.5 mM EGTA, 2 mM ADP, 50 mM KCl, 0.2 mg/ml bovine serum albumin, 20 µM Ap5A,1 5 µmol/min hexokinase, 1 mM glucose, 1 mM dithiothreitol, 5 mM MgCl2, and 0.01% NaN3. Regulated thin filaments were prepared by adding 0.35 µM tropomyosin and 0.35 µM troponin, enough to ensure saturation of the actin. For experiments done in the presence of Ca2+, 0.6 mM CaCl2 was added. Using a SLM 8000 spectrofluorometer outfitted with a stirred water jacket sample holder, 1.8-ml samples were excited at 368 nm, and the emission intensity was monitored at 407 nm.

Electron Micrographs-- Ca2+ G-actin was incubated in 2 mM EGTA, 1 mM MgCl2 to convert it to the Mg2+ form (30) and then polymerized by the addition of 3 mM MgCl2 and equimolar phalloidin. F-actin was then diluted in 100 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, 5 mM NaH2PO4 (pH 7.0), 5 mM Pipes (pH 7.0), 1 mM NaN3, applied to thin carbon-coated electron micrograph grids, and negatively stained with 1% uranyl acetate (35). Electron micrograph images were recorded at ×60,000 magnification under low dose conditions (~12 e-/Å2) on a Philips CM120 electron microscope. Micrographs were digitized using either Imacon Flextight Precision II or Zeiss SCAI scanners at a pixel size corresponding to ~7.0 Å in the filaments (36). Regions of filaments suitable for helical reconstruction were selected on the basis of filament straightness and lack of aggregation, uniformity of staining, and freedom from astigmatism. Slightly curved filaments were straightened by applying spline fitting algorithms (37). Helical reconstruction was carried out using standard methods (38-40) as described previously (8, 36).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tropomyosin Has Increased Affinity for D56A/E57A Actin Compared with Wild Type Actin-- Previous experiments (16, 17, 19) suggest that modification or mutation of actin subdomain 2 can alter interactions of actin with tropomyosin (16). To explore this quantitatively, we examined the binding of tropomyosin to yeast actin mutant D56A/E57A (Fig. 1). The structure of subdomain 2 may depend on whether Ca2+ or Mg2+ is bound in the actin binding cleft (30, 41), so we focused the study on tropomyosin binding to Mg2+ F-actin, containing the physiologically relevant metal. To produce Mg2+ F-actin, Ca2+ bound to wild type, and mutant G-actin was exchanged for Mg2+ prior to polymerization. Using this actin, there was a 3-fold increase in the affinity of tropomyosin for D56A/E57A actin (*) as compared with wild type F-actin (*) (Kapp = 4.9 ± 0.2 × 106 M-1 versus Kapp = 1.62 ± 0.04 × 106 M-1, Fig. 1A). Kapp partially depends upon cooperative aspects of binding (see "Materials and Methods"). However, this increased affinity cannot be explained by greater tropomyosin-tropomyosin cooperativity in the presence of the mutation, because tropomyosin binding was less cooperative to mutant actin than to control actin (y = 35 ± 7 versus y = 134 ± 34). This strongly suggests that the mutation increased tropomyosin's actin affinity per se.


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  		Fig. 1.   Altered tropomyosin binding to mutant yeast F-actin. A, tropomyosin binding to Mg2+ F-actin. Illustrated composite data from several experiments and best fit binding curves show that tropomyosin bound more strongly to D56A/E57A actin (*, Kapp = 4.9 ± 0.2 × 106 M-1 and y = 35 ± 7) than to wild type yeast actin (open circle , Kapp = 1.62 ± 0.04 × 106 M-1 and y = 134 ± 34). B, tropomyosin binding to conventionally polymerized F-actin, in the presence and absence of 5 µM myosin S1. Unlike A, Ca2+ G-actin was not converted to Mg2+ G-actin prior to initiation of polymerization. The results were more variable than in A, but again showed higher tropomyosin affinity for mutant actin (*, Kapp = 2.6 ± 0.2 × 106 M-1 and y = 19 ± 6) than for wild type actin (open circle , Kapp = 1.8 ± 0.1 × 106 M-1 and y = 57 ± 9). In the presence of myosin S1, tropomyosin bound very tightly to both wild type yeast actin (triangle ) and D56A/E57A actin (+) with Kapp >=  9 × 106 M-1. Conditions: 5 µM phalloidin stabilized Mg2+ F-actin, 10 mM Tris, pH 7.5, 5 mM MgCl2, 100 mM KCl, 0.1 mM dithiothreitol, and 1 mM EGTA.

When actin was polymerized without prior exchange of metals, the binding data were much more variable (Fig. 1B, *, ). This may reflect heterogeneity in F-actin structure due to the Ca2+/Mg2+ mixture (41), but this was not assessed in the present work. Despite the variability in the data, the mutant actin again bound to tropomyosin more tightly. Another important condition to evaluate was the interaction of tropomyosin with actin filaments decorated with myosin heads. In the presence of myosin S1 tropomyosin binding to yeast or muscle actin was nearly stoichiometric and difficult to measure (21, 24). This was also true for D56A/E57A actin (Fig. 1B, +) and was confirmed for control yeast actin (Delta ).

Affinity of the Regulatory Complex for Actin Was Unaffected by the Actin Mutation D56A/E57A-- Because the affinity of tropomyosin was increased for D56A/E57A F-actin, we examined whether the affinity of the troponin-tropomyosin complex was also altered for the mutant. In the absence of Ca2+ (Fig. 2A) the regulatory complex bound equally tightly to both the wild type (triangle ) and mutant (+) F-actins (Kapp = 8.1 ± 0.2 × 106 M-1 and Kapp = 8.1 ± 0.4 × 106 M-1, respectively). Because the binding of the tropomyosin-troponin complex is tight, it was possible that a change in the affinity of the complex for the mutant actin might not be apparent. Therefore, the KCl concentration was increased from 300 to 400 mM to weaken binding. This achieved the desired effect on affinity, but for unknown reasons it also produced much more scatter in the data (lower two curves in Fig. 2A). With this limitation, the affinities of the regulatory complex for mutant (*) and wild type (black-square) F-actins were not distinguishable (3.2 ± 0.2 and 3.5 ± 0.3 × 106 M-1, respectively).


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  		Fig. 2.   Unaffected tropomyosin-troponin binding to mutant actin. A, tropomyosin-troponin binding to yeast F-actin in the absence of Ca2+. In the presence of 300 mM KCl, the regulatory complex bound with indistinguishable affinities to wild type yeast actin (triangle ) and mutant actin (+). Kapp = 8.1 ± 0.2 × 106 M-1 and y = 45 ± 9 for control actin and Kapp = 8.1 ± 0.4 × 106 M-1 and y = 33 ± 9 for mutant actin. The mutation also had no affect on affinity in the presence of 400 mM KCl, with Kapp = 3.5 ± 0.3 × 106 M-1 and y = 19 ± 7 for wild type actin (open circle ), and Kapp = 3.2 ± 0.2 × 106 M-1 and y = 41 ± 3 for mutant actin (*). B, tropomyosin-troponin binding to yeast F-actin in the presence of Ca2+. The wild type data (open circle ) imply Kapp = 4.84 ± 0.09 × 106 M-1 and y = 58 ± 7, and the mutant actin (*) data indicate slightly weaker Kapp = 3.5 ± 0.2 × 106 M-1 and y = 38 ± 9. The figure is a composite of three or more data sets under each condition, and the solid lines are best fit theoretical curves. Conditions: 10 mM Tris (pH 7.5), 3 mM MgCl2, 2 mM dithiothreitol, 0.3 mg/ml bovine serum albumin, 0.5 mM EGTA (± 0.6 mM CaCl2), and either 7 µM phalloidin-treated yeast F-actin in the presence of 300 mM KCl or 5 µM phalloidin-treated yeast F-actin in the presence of 400 mM KCl.

Ca2+ binding to troponin decreased by about 50% the affinity of the regulatory proteins for both wild type and mutant actins in the presence of 300 mM KCl. As shown in Fig. 2B, the affinity was slightly weaker for D56A/E57A F-actin (*, Kapp = 3.5 ± 0.3 × 106 M-1) than for wild type F-actin (, Kapp 4.84 ± 0.09 × 106 M-1). This is only a 1.4-fold effect, and its significance is not clear.

Yeast Actin Mutant D56A/E57A Shows Altered Myosin S1 ATPase Activity-- Because D56A/E57A F-actin bound to the regulatory proteins with nearly normal affinity in both the presence and absence of Ca2+, one might predict that regulated thin filaments composed of the mutant actin would undergo Ca2+-dependent regulation of myosin S1 MgATPase activity. Table I shows the results of experiments to test this. Both Mg2+ F-actin and conventionally polymerized actin were examined. The myosin S1-unregulated actin MgATPase rates were decreased by 65-80% as a result of the mutation. The actin mutation decreased the myosin S1 MgATPase rate for actin-tropomyosin moderately, by 45%, but for the actin-tropomyosin-troponin in the presence of Ca2+ the mutation decreased the rate by 88-94%. Regulated thin filaments containing wild type actin showed a 3-5-fold increase in ATPase rates upon the addition of Ca2+. Notably, regulated thin filaments composed of the D56A/E57A F-actin failed to exhibit this activation, showing an increase upon Ca2+ addition of only 20%. In the absence of Ca2+, troponin-tropomyosin inhibited the actin-myosin S1 MgATPase rate for both wild type actin and mutant actin. The exchange of Ca2+ for Mg2+ on G-actin prior to polymerization had no effect on these comparisons.

                              
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Table I
Effect of D56A/E57A actin mutation on actin-activated myosin S1 MgATPase rates
ATPase assay conditions were as described under "Materials and Methods." Averages are from four or more independent experiments. Conventional F-actin was prepared by adding MgCl2 and KCl to Ca2+ G-actin. Mg2+ F-actin was prepared by exchanging Mg2+ for Ca2+ bound to G-actin, prior to polymerization. ATPase results did not significantly differ for conventional versus Mg2+ F-actins.

Yeast Actin Mutant D56A/E57A Has Altered Myosin S1 Affinity-- Actin-activated myosin S1 MgATPase rates were decreased for the mutant actin, suggesting that myosin S1 binding might be altered by the mutation. As previously shown (34) myosin S1-ADP binding to pyrene-labeled actin produces a decrease in fluorescence intensity, from which the affinity constant for unregulated pyrene muscle actin under the current conditions was determined (open circle , Fig. 3A, and Table II). This measurement was combined with a competitive binding assay (Fig. 3, B and C) to determine the myosin S1 affinity for both yeast actins, as well as for unlabeled skeletal muscle actin, which was used as a control to allow comparison of unlabeled yeast and muscle actins. Similarly to results seen in the actin-activated myosin S1 MgATPase assays, the D56A/E57A mutation decreased the affinity of myosin S1 for actin ~3-fold (Table II and triangles versus squares, Fig. 3B). In agreement with published comparisons (42), myosin S1-ADP had ~10-fold lower affinity for wild type yeast actin than for unlabeled rabbit muscle actin (Fig. 3B, squares versus circles). Also, the data show that pyrene labeling decreased the affinity of myosin S1-ADP for muscle actin approximately 5-fold, from 17 to 3.4 × 106 M-1.


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  		Fig. 3.   Myosin S1-ADP binding to F-actin and to regulated thin filaments. The panels show normalized alterations in pyrene actin fluorescence intensity as the amount of myosin bound to the filament changes, with zero corresponding to myosin-saturated filaments. A, noncompetitive binding titration of myosin S1-ADP added to pyrene-labeled rabbit fast skeletal muscle F-actin in the presence () and absence (open circle ) of troponin-tropomyosin. Actin-myosin S1 affinity was increased by the regulatory proteins (see Table II) and was sufficiently tight so that cooperativity was not observed, and results were similar in the presence and absence of Ca2+ (both are shown by ). B, competitive displacement of myosin S1 from pyrene-labeled muscle actin by the addition of unlabeled actins. Each data point represents duplicate data sets that were normalized and then averaged. The solid lines represent the best fit of the data to Equation 2. Unlabeled, unregulated rabbit fast skeletal muscle actin () bound myosin S1-ADP much more tightly than did wild type yeast actin (black-square), a 12-fold effect (Table II). The D56A/E57A mutation (black-triangle) decreased the affinity of myosin S1 further, an additional 2.6-fold difference. C, competitive binding of myosin S1 to pyrene-labeled versus unlabeled actin-troponin-tropomyosin filaments. Filled symbols represent experiments done in the presence of Ca2+, open symbols represent experiments done in the absence of Ca2+, and solid lines are best fit curves using Equation 2. Data for muscle actin thin filaments in the presence () and absence (open circle ) of Ca2+ were similar and were fit together. For thin filaments containing wild type yeast actin (black-square), myosin S1-ADP binding was much weaker than for muscle actin in the presence of Ca2+, a 13-fold difference, and binding was much weaker still in the absence of Ca2+ (), an additional 18-fold difference (Table II). Most importantly, the D56A/E57A mutation (black-triangle) weakened by 13-fold the affinity of myosin S1-ADP for yeast actin-regulated thin filaments in the presence of Ca2+. The dashed line represents the expected behavior for D56A/E57A-regulated filaments if the mutation had only a 2.6-fold effect, as it does in the absence of troponin-tropomyosin (B).

                              
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Table II
Thin filament-myosin S1 affinities for various actins
For pyrene-labeled skeletal muscle actin the affinities were measured by the sequential addition of myosin S1 to thin filaments labeled with pyrene-iodoacetamide on actin Cys374, causing progressive quenching of fluorescence intensity. All other affinities were determined by sequential addition of nonlabeled thin filaments, resulting in competitive displacement of myosin S1 from pyrene-labeled muscle actin and a progressive increase in fluorescence intensity. Conditions are described under "Materials and Methods."

Actin-myosin S1 binding was also examined in the presence of troponin-tropomyosin. Unlabeled rabbit muscle actin (Fig. 3C, circles) again showed a 5-fold increase in affinity (Table II) as compared with pyrene-labeled actin (Fig. 3A, filled circles). The fluorescence titrations for unlabeled muscle actin were similar in either the absence or presence of Ca2+ (Fig. 3C, open versus filled circles). Therefore curve fitting was performed on combined data from both conditions. In contrast, myosin S1 binding to wild type yeast actin-tropomyosin-troponin was much weaker in the absence than in the presence of Ca2+ (open versus filled squares). Furthermore, even in the presence of Ca2+, myosin S1 bound much more weakly to regulated thin filaments containing yeast rather than muscle actin. The affinity of myosin S1 for muscle actin filaments was 13-fold higher than its affinity for wild type yeast actin filaments (filled circles versus squares, 95 versus 7.3 × 106 M-1). This is similar to what was found in the absence of the regulatory proteins, above.

In the presence of Ca2+, myosin S1 showed much lower affinity for D56A/E57A F-actin-tropomyosin-troponin than for wild type filaments (filled triangles versus filled squares, Fig. 3C), 7.3 × 106 versus 5.6 × 105 M-1. This effect of the mutation on thin filament-myosin S1 binding was much greater in the presence of tropomyosin-troponin-Ca2+ than for bare actin. The dashed line in Fig. 3C shows the expected binding curve if the myosin affinity were weakened by the same amount as for bare actin, a 2.6-fold effect. Instead, the mutation caused much greater weakening (black-triangle), an estimated 13-fold effect.

In the absence of Ca2+, myosin S1 was only minimally displaced from labeled muscle thin filaments by the addition of yeast thin filaments, either wild type or mutant (open squares and triangles). Removal of Ca2+ decreased the myosin S1 thin filament affinity ~18-fold. This weak binding to control yeast actin filaments made it impossible to assess the effects of the mutation on myosin affinity for actin-troponin-tropomyosin in the absence of Ca2+.

Electron Microscopy of Thin Filaments Containing D56A/E57A Actin-- Electron microscopy was performed to distinguish possible structural reasons for the properties of D56A/E57A F-actin. Electron micrographs of negatively stained wild type actin yielded individual extended filaments (Fig. 4A), whereas mutant actin was bundled into large laterally associated aggregates (Fig. 4C). This is similar to findings when negative charges are deleted from a different region of actin, the NH2 terminus (29). In many systems, increasing ionic strength reduces F-actin bundle formation caused by actin-associated binding proteins. However, in our case neither the addition of 400 mM NaCl to the mutant F-actin nor polymerization of the D56A/E57A actin in the presence 300 mM NaCl caused an apparent reduction in bundling.


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  		Fig. 4.   Electron micrographs and three-dimensional reconstructions of thin filaments. Representative micrographs of negatively stained phalloidin-stabilized wild type (A and B) and D56A/E57A mutant (C and D) yeast Mg2+ F-actin, (A and C) F-actin alone, (B and D) F-actin-tropomyosin complexes. Note dispersed single filaments in A, B, and D and bundled ones in C. Scale bar represents 200 nm. Experimental conditions are given under "Material and Methods." Image reconstruction was carried out on 15 Ca2+-treated mutant filaments containing tropomyosin and troponin (E and F). E, helical projection and (F) transverse section of maps of three-dimensional reconstructions showing tropomyosin (arrows) positioned on the outer aspect of the inner domain of actin, i.e. the same Ca2+-induced position found in control filaments treated with Ca2+ (43, 44); subdomains 1-4 are labeled. Helical projections were formed by projecting component densities down the long pitch actin helices (i.e. along the n = 2 helical tracks) onto a plane perpendicular to the thin filament axis; hence, the resulting projections show axially averaged positions of tropomyosin relative to actin made bilaterally symmetric. In contrast, the transverse sections show the position of tropomyosin at a given level along filaments and connectivity to specific subdomains of actin. Because adjacent actin monomers on either side of filament axis are staggered, sectioning through the center of actin subdomains-1 and -3 of one actin monomer results in sectioning through subdomains-2 and -4 of the other.

In marked contrast, the addition of tropomyosin prevented bundling of the mutant actin, and single filaments were observed for both wild type and mutant actins (Fig. 4, B and D). This made it possible to carry out three-dimensional image reconstruction to analyze the structure of D56A/E57A actin-troponin-tropomyosin. The position of tropomyosin was easily identified in helical projection or cross-sections of such reconstructed filaments (Fig. 4, E-F). In filaments examined in the presence of Ca2+, tropomyosin was located at the outer edge of the inner domain of actin, exposing most of the actin residues believed to interact with myosin. This result is indistinguishable from that observed in the presence of Ca2+ for regulated muscle thin filaments (3, 43). Because tropomyosin is found in the normal Ca2+-induced position, the inhibitory effects of the actin mutation on myosin S1 thin filament binding and on MgATPase rate activation were not because of tropomyosin adopting a blocking position on the actin outer domain as normally occurs in the absence of Ca2+.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The interactions of troponin, tropomyosin, and myosin with actin are central to muscle contraction and its regulation but are incompletely understood. High resolution structures are not available for the component proteins when contacting each other. Furthermore, the dynamic nature of the system presents a challenging complexity. A major unsolved problem is to determine the specific interactions between actin and these other proteins under different conditions. The present report addresses this subject by presenting several unexpected properties of thin filaments containing actin subdomain 2 mutation D56A/E57A. The results suggest unanticipated roles for this region in actin binding to myosin, tropomyosin, and possibly troponin. In addition, the data support the conclusion (12, 44) that Ca2+-induced shifting of tropomyosin on actin is insufficient for activation of myosin cycling and further suggest that Ca2+-troponin-tropomyosin is inhibitory unless myosin-actin binding is sufficiently strong.

The D56A/E57A mutation does not directly involve actin residues that bind to myosin (45). Nevertheless, it weakens the affinity of myosin S1 for unregulated actin almost 3-fold and similarly decreases the actin-myosin S1 MgATPase rate. Two possible explanations should be considered. One explanation is that these D56A/E57A effects primarily are a consequence of actin filament bundling, with resultant inaccessibility of many actin monomers. If this is true, then one might also expect tropomyosin binding to be weakened, but instead it is increased 3-fold by the mutation. A second possibility, more likely in our view, is that these results are supporting structural and kinetic data demonstrating interactions between myosin and actin subdomain 2 (25, 46, 47), and it is these interactions that are inhibited by the mutation. In favor of this, both myosin binding to actin and the MgATPase rate remain suppressed by the mutation when troponin-tropomyosin and calcium are added, despite the absence of bundling. We suggest that this is caused by the same reduction in myosin binding observed in the absence of troponin-tropomyosin but that the decrease in actin-myosin affinity is exaggerated by the cooperativity-inducing regulatory proteins. Myosin binding to the regulated thin filament requires cooperative tropomyosin movement onto the actin inner domain (8). Initiation of this movement may require myosin binding of a certain strength. Furthermore, myosin binding induces conformational changes in actin (48-50) that are of proposed importance for tropomyosin movement onto the actin inner domain and relief of inhibition (44).2 These changes in actin are not yet defined but could be altered by the D56A/E57A mutation.

The current ATPase results fit a pattern indicating that troponin-tropomyosin-mediated regulation is impaired by any one of a variety of structural changes in subdomain 2 (17, 19). The present work suggests that these functional defects can occur with preserved affinity of the regulatory proteins for actin and, at least in the present case, may best be understood as consequences of altered affinity of myosin for actin. Relief of the inhibitory effects of troponin-tropomyosin requires not just Ca2+ binding to troponin but also strong myosin binding to the thin filament (11). Subdomain 2 may play an important role in myosin binding and myosin-induced activation, which is suppressed by the D56A/E57A mutation. Previous structural studies have implicated actin subdomain 2, albeit not residues Asp56/Glu57 themselves, in myosin binding to actin (25, 46, 47). Considering the relatively dynamic properties of this subdomain (30, 41, 52, 53), it is plausible to suggest that mutation of residues 56 and 57 affects myosin binding via indirect changes in actin subdomain 2, rather than by directly altering myosin-actin interactions.

Structural studies of actin-troponin-tropomyosin in the off state (i.e. in the absence of Ca2+) indicate that tropomyosin may bridge over subdomain 2 without extensive contact (4). Yet, studies that determine contact by chemical modification and cross-linking raise the possibility that tropomyosin makes direct contact with subdomain 2 residues Lys50, Lys61, and Arg95 (17-20). Actin mutants provide an alternative approach to assess specific interactions between tropomyosin and actin. In one such study, tropomyosin suppressed the in vitro motility of E93K actin filaments, leading to the suggestion that tropomyosin binding to the outer domain of actin is affected by charged surface residues on subdomain 2 (16). The present D56A/E57A binding data support the conclusion that acidic residues on subdomain 2 normally act to weaken tropomyosin binding.

On the other hand, the present structural data suggest that the inhibitory properties of subdomain 2 modifications are not because of tethering of tropomyosin on the actin outer domain, as inferred previously (16). Ca2+ causes tropomyosin to shift normally on mutant actin filaments, exposing most of the myosin binding sites on actin, but thin filament activation remains inhibited by the actin mutation. These results emphasize the critical importance of the additional movement of tropomyosin further onto the inner actin domain, a movement observed when myosin S1 is bound to actin. Weakened myosin binding caused by the D56A/E57A mutation may impair this additional tropomyosin movement, and a similar mechanism may occur for the E93K mutation, which also weakens myosin binding to actin according to a recent report (47). Finally, the present results closely parallel those for muscle actin-troponin-tropomyosin filaments containing an inhibitory, deletion mutant tropomyosin (44). These filaments undergo normal Ca2+-induced movement of tropomyosin on actin, but myosin binding and cycling are inhibited by impairment of the myosin-induced additional shift in tropomyosin position further onto the actin inner domain.

In the presence of troponin, the binding of the regulatory complex is minimally affected by the D56A/E57A mutation. This suggests either that tropomyosin-troponin does not interact with residues 56 and 57 or that the mutation has counter-balancing effects, strengthened tropomyosin-actin interactions as observed in the absence of troponin (a 3-fold effect, Fig. 1) but correspondingly weakened troponin-actin interactions. In regard to this latter possibility, basic portions of troponin I and of the COOH-terminal portion of troponin T might have decreased interactions with actin that has been rendered less acidic by the D56A/E57A mutation. The importance of such interactions is suggested by a preliminary reconstruction of troponin on regulated filaments showing that troponin may contact the surface of actin subdomain 2 in the absence of Ca2+ (51).

In conclusion, regulated thin filaments containing troponin, tropomyosin, and D56A/E57A actin did not exhibit Ca2+-mediated activation of myosin S1 MgATPase rates. Correspondingly, myosin S1 exhibited much lower affinity for mutant than for wild type-regulated thin filaments. The mutation more modestly decreased the affinity of myosin S1 for unregulated actin, as well as the MgATPase rate in the absence of troponin and tropomyosin. Despite the inhibitory properties of Ca2+-troponin-tropomyosin-mutant actin filaments, Ca2+ caused tropomyosin to shift toward the actin inner domain, exposing most but not all of the actin sites that bind to myosin, similar to the tropomyosin shift that occurs for muscle actin filaments. The results illustrate the interconnection between myosin binding and thin filament activation and suggest the importance of charged actin subdomain 2 surface residues for both myosin binding to actin and conformational switching of the thin filament.

    ACKNOWLEDGEMENTS

We thank Dr. David Drubin for the gift of the D56A/E57A yeast actin strain.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL-38834 (to L. S. T.), HL-36153 (to W. L.), AR-34711 (R. C.), the Shared Instrumentation Grant RR08426 (R. C.), and AHA IA-97-SA-3 (V. L. K.).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.

** To whom correspondence should be addressed: The University of Iowa, Dept. of Internal Medicine, 200 Hawkins Dr., SE610, GH, Iowa City, IA 52242. Tel.: 319-356-3703; Fax: 319-356-3086; E-mail: larry-tobacman@uiowa.edu.

Published, JBC Papers in Press, May 5, 2000, DOI 10.1074/jbc.M002939200

2 L. S. Tobacman and C. A. Butters, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: Ap5A, P1,P5-di(adenosine 5')-pentaphosphate; Pipes, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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