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J Biol Chem, Vol. 274, Issue 44, 31279-31285, October 29, 1999


Effects of Tropomyosin Internal Deletions on Thin Filament Function*

Cheryl LandisDagger , Nick Back§, Earl Homsher§, and Larry S. TobacmanDagger parallel

From the Departments of Dagger  Internal Medicine and  Biochemistry, the University of Iowa, Iowa City, Iowa 52242 and the § Department of Physiology, UCLA, Los Angeles, California 90024

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Striated muscle tropomyosin spans seven actin monomers and contains seven quasi-repeating regions with loose sequence similarity. Each region contains a hypothesized actin binding motif. To examine the functions of these regions, full-length tropomyosin was compared with tropomyosin internal deletion mutants spanning either five or four actins. Actin-troponin-tropomyosin filaments lacking tropomyosin regions 2-3 exhibited calcium-sensitive regulation in in vitro motility and myosin S1 ATP hydrolysis experiments, similar to filaments with full-length tropomyosin. In contrast, filaments lacking tropomyosin regions 3-4 were inhibitory to these myosin functions. Deletion of regions 2-4, 3-5, or 4-6 had little effect on tropomyosin binding to actin in the presence of troponin or troponin-Ca2+, or in the absence of troponin. However, all of these mutants inhibited myosin cycling. Deletion of the quasi-repeating regions diminished the prominent effect of myosin S1 on tropomyosin-actin binding. Interruption of this cooperative, myosin-tropomyosin interaction was least severe for the mutant lacking regions 2-3 and therefore correlated with inhibition of myosin cycling. Regions 3, 4, and 5 each contributed about 1.5 kcal/mol to this process, whereas regions 2 and 6 contributed much less. We suggest that a myosin-induced conformational change in actin facilitates the azimuthal repositioning of tropomyosin which is an essential part of regulation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiac and skeletal muscle contractions are regulated by calcium- and myosin-induced alterations in the conformation of the thin filament (1-3). An important component of these conformational changes is believed to be azimuthal repositioning of tropomyosin, which unblocks sites on actin which bind to myosin. (A dissenting viewpoint is presented in Ref. 4.) Recent structural studies indicate that most of this unblocking occurs when calcium binds to troponin-tropomyosin but that the myosin binding site is not fully exposed in the calcium conformation of the thin filament (5-7). Several functional changes in the thin filament occur specifically when myosin is bound (8-17), and recent structural data show that in this circumstance tropomyosin undergoes a smaller, additional azimuthal movement (5). This results in full exposure of the myosin binding site on actin. The mechanisms for these tropomyosin movements are poorly understood.

Examination of tropomyosin mutants has been helpful in understanding the regulatory function of the thin filament. In particular, rat alpha -Tm(Delta 47-165) is a recombinant tropomyosin that is missing 119 internal residues, which spans four instead of the usual seven actins, and which inhibits both in vitro motility and solution myosin S11 MgATPase activity (8). This tropomyosin (here redesignated Delta 234Tm because it lacks the second, third, and fourth of tropomyosin's seven quasi-repeats) bound much more weakly to myosin S1-decorated actin than did control tropomyosin. Therefore, it was suggested that its inhibitory properties were caused by destabilization of the myosin-induced conformation of the thin filament, the conformation corresponding to full azimuthal movement of tropomyosin. This conclusion has since been supported by three-dimensional reconstructions of negatively stained thin filament electron micrographs, which demonstrate that the tropomyosin deletion does not alter the large, calcium-induced azimuthal movement of tropomyosin.2 The results suggested that the calcium-induced movement of tropomyosin is insufficient to permit cross-bridge function. Rather, full tropomyosin movement is required.

These previous data suggest that the deleted portion of tropomyosin is somehow important for thin filament activation, but they do not identify the specific structural changes in Delta 234Tm which are responsible for its altered function. The mutation's dual effects of impaired tropomyosin binding to actin-S1 and inhibition of myosin cycling could be coincidental properties of one mutant or could suggest more general features that are necessary for proper thin filament regulation. Also unclear is whether the most significant feature of Delta 234Tm is its shortened length, or whether its behavior is caused by deletion of a specific region. To explore these issues, we now report the properties of a series of tropomyosin internal deletion mutants. The results indicate that it is not the length of tropomyosin that is critical for proper regulatory function, but rather it is the specific regions of tropomyosin which are present or missing. Also, a broad internal region of tropomyosin is important for tropomyosin binding to myosin S1-decorated actin, and the strength of this binding process correlates with retention of physiological troponin-tropomyosin-mediated regulation.

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

Preparation of Tropomyosins Containing Internal Deletions-- Rat striated/cardiac muscle alpha -tropomyosin cDNA (19) was altered using the ExSite mutagenesis kit (Stratagene). This approach uses polymerase chain reaction and contiguous oppositely directed primers to generate circular plasmids missing a region that is a gap in one of the primers. The starting DNA encoded Met-Ala-Ser-tropomyosin in pET3d (8). All mutant coding sequences were confirmed by automated sequencing at the University of Iowa DNA Core Facility. The mutant constructs included deletions of either 231 base pairs (Delta 23Tm and Delta 34Tm) or 357 base pairs (Delta 345Tm and Delta 456Tm). Numbering according to the rat sequence, the deletions were: Gln47 through Ser123 (Delta 23Tm), Asn89 through Val165 (Delta 34Tm), Asn89 through Leu207 (Delta 345Tm), and Glu124 through Ala242 (Delta 456Tm). Using this same designation pattern, previously described Delta 234Tm lacks residues Gln47 through Val165. (The previous notation ralpha -Tm Delta (49-167) took the beginning of the fusion dipeptide Ala-Ser as residue 1.)

Protein Purification-- Recombinant tropomyosins were purified according to Willadsen et al. (20). Rabbit fast skeletal muscle actin (21) and myosin subfragment 1 (22) and bovine cardiac troponin (23) were obtained using published procedures. Prior to motility assays, rabbit skeletal muscle heavy meromyosin was mixed with actin in the presence of ATP, and inappropriately strong binding molecules were removed by centrifugation (24). Cardiac troponin subunits were purified as reported by Tobacman and Lee (25), and the TnT subunit was stoichiometrically labeled on Cys39 with [3H]iodoacetic acid (26). The labeled TnT was then mixed in a 1:1:1 ratio with the other troponin subunits under denaturing, high ionic strength conditions, and the troponin complex was purified as described (25). Tropomyosin was labeled on Cys190 under denaturing conditions with [3H]iodoacetic acid (27).

Assays-- The binding of regulatory proteins to actin was measured by cosedimentation in a TLA100 rotor (Beckman) for 30 min at 35,000 rpm after a 30-min incubation at 25 °C. Conditions were: 10 mM NaH2PO4 (pH 7.0), 5 mM MgCl2, 1 mM EGTA, 4.5 or 5 µM actin, either 0 or 5 µM myosin S1, and between 60 and 300 mM KCl (see figure legends). Total and supernatant radioactivity were compared to calculate the bound protein. When troponin as well as tropomyosin was present, either tropomyosin was added in a 0.1 µM excess over troponin when the label was on the troponin, or troponin was added in the same excess when the label was on the tropomyosin. Binding data were fit initially with MATLAB (The Math Works), using an equation for the binding of a long ligand to a linear lattice (27-29). Different data sets were then normalized based upon the MATLAB results, similar experiments combined, and the program Scientist (MicroMath) used for refinement and for calculation of errors. Competitive binding experiments were done under the same conditions as above, except an identical saturating concentration (see legends) of [3H]Delta 234Tm was included in all samples. The competition was analyzed as reported previously for competitive binding of troponin to the thin filament (30). Curvefitting with Scientist was used to determine the ratio of the actin or actin-S1 affinities of the competing unlabeled tropomyosin versus the labeled tropomyosin.

In vitro sliding of filaments over a heavy meromyosin-coated coverslip was examined by epifluorescence microscopy and digital analysis of movement (31). Rhodamine phalloidin-labeled actin was bound to the motility surface in the absence of ATP, and then 100 nM troponin was added, along with 100-150 nM of one of the various forms of tropomyosin. When ATP was added movement was assessed both in the presence (pCa 5) and in the absence (pCa 9) of calcium and at ionic strengths of 50 and 100 mM.

MgATPase assays were performed as described previously (32), under conditions of low myosin S1:actin ratio and saturation, and linearity with myosin S1 concentration: 10 mM NaH2PO4 (pH 7.0), 5 mM MgCl2, 1 mM [gamma -32P]ATP, 4 µM actin, 1 µM myosin S1, and either 0.5 mM EGTA or 0.1 mM CaCl2. Troponin and tropomyosin concentrations are indicated in the figure legends.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Design of a Series of Tropomyosin Internal Deletion Mutants-- Fig. 1 schematically shows the alpha -striated muscle tropomyosins examined in the present report. The tropomyosin amino acid sequence has seven quasi-repeating regions with periodicity corresponding to the spacing between adjacent actin residues on the filament (33). The first residues of the regions are defined for the present study as indicated beneath the full-length molecule in Fig. 1. Paramount in these definitions was the constraint that the number of residues deleted should be a multiple of seven, to preserve the heptad repeat of the coiled-coil. Also, the boundaries used in previous studies (8, 34, 35) of regions 2, 3, and 4 were retained. Mutant tropomyosins either spanned five actins as a result of the deletion of 77 residues (two regions), or spanned four actins as a result of the deletion of 119 residues (three regions). ASTm and Delta 234Tm are the same molecules studied previously (8). All forms include an Ala-Ser NH2-terminal dipeptide that corrects the poor polymerizability of unacetylated tropomyosin expressed in bacteria (36).


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  		Fig. 1.   Schematic representation of rat alpha -striated muscle tropomyosin internal deletion mutants. Recombinant tropomyosin cDNAs were designed for bacterial expression with, after processing, an Ala-Ser NH2-terminal dipeptide that corrects the poor polymerizability of unacetylated tropomyosin (36). The first residue of each similar, quasi-repeating region (33) is identified beneath the full-length molecule. Regions retained in each mutant are indicated. Tropomyosins spanning five actins have 77 residues missing, and those spanning four actins have 119 residues deleted. Note that troponin binds most tightly near the COOH terminus of tropomyosin (region 7) but also stretches over regions 6, 5, and possibly part of region 4.

Troponin binds to tropomyosin most tightly by interactions between the NH2-terminal portion of TnT and the COOH terminus of tropomyosin (region 7) (30, 37-42). There is a weak site of troponin binding near tropomyosin residues 150-190 in repeating regions 4 and 5 (38, 40, 43-46), and the two proteins may be in contact for an extended region involving the COOH-terminal third of tropomyosin (47). Therefore, constructs lacking regions 4, 5, and/or 6 may have altered interactions with troponin. However, the strongest site of interaction (region 7) is retained in all mutants, as are sequences involved in normal end-to-end tropomyosin-tropomyosin contacts.

Effect of Internal Tropomyosin Mutations on Calcium-mediated Regulation-- The effects of the mutations on the unloaded sliding of heavy meromyosin-propelled actin-troponin-tropomyosin thin filaments were examined in an in vitro motility assay (31). In the presence of calcium 90% of the thin filaments containing full-length cardiac tropomyosin moved at a fast uniform sliding speed (right half of Table I). Removal of calcium inhibited the sliding of 97% of these control filaments, and those that continued to move smoothly did so at <7% of the speed observed in the presence of calcium. Filaments containing bovine cardiac tropomyosin or recombinant full-length ASTm behave indistinguishably (8). Delta 23Tm also caused calcium-sensitive movement, although the regulation was not as complete: 67% of the filaments moved continuously when calcium was added, at 73% of the speed of the control filaments. Delta 34Tm is the same length as Delta 23Tm but had very different effects: few filaments moved in the presence or in the absence of calcium, and those that moved continuously did so with <7% of the speed of the control filaments. Delta 345Tm and Delta 456Tm filaments were also poorly regulated, with movement greatly suppressed in the presence of calcium as well as in its absence. Comparable results were obtained when the ionic strength was decreased from 100 mM to 50 mM (data not shown). All of the troponin-tropomyosin complexes suppressed movement in the absence of calcium, and calcium relieved the inhibition only for control filaments and for Delta 23Tm filaments.

                              
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Table I
Effect of tropomyosin internal deletions on regulated movement in motility assays
Assay conditions were: temperature, 25 °C; ionic strength adjusted to 100 mM with MOPS buffer at pH 7.4; MgCl2, 2, Na2ATP, 1 mM; EGTA, 2 mM. 0.4% methyl cellulose was added to the motility solution to hold filaments to the motility surface. The motility solution contained 100-150 nM bovine cardiac tropomyosin or mutant tropomyosin and 100 nM troponin. A uniformly moving filament was defined as one with time point to time point speeds exhibiting a S.D. less than half the mean speed for that filament.

The effects of the mutations were also examined in myosin S1 MgATPase assays, and the results (Fig. 2) paralleled the motility data. Delta 23Tm filaments (×, EGTA; *, Ca2+) displayed calcium-sensitive ATPase rate regulation, similar to data reported for ASTm (8). In contrast, actin-troponin-tropomyosin filaments containing Delta 234Tm, Delta 345Tm, and Delta 456Tm, and Delta 34Tm were all inhibitory to the ATPase rate, both in the presence (filled symbols) and in the absence (open symbols) of calcium (Fig. 2). Filaments containing Delta 456Tm (triangles) demonstrated slightly less inhibition than the others, similar to the motility data (Table I).


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  		Fig. 2.   Effect of internal tropomyosin deletions on Ca2+-mediated regulation of the myosin S1-thin filament MgATPase rate. Increasing concentrations of various troponin-tropomyosin complexes were added to samples with unchanging concentrations of actin and myosin S1. Data points are offset slightly for clarity. Averaged rates from two experiments are shown. Open symbols and dashed lines, 0.5 mM EGTA. Filled symbols and solid lines, 0.1 mM CaCl2. Circles, Delta 234Tm; squares, Delta 345Tm; triangles, Delta 456Tm; crosses, Delta 23Tm; diamonds, Delta 34Tm. Troponin-Delta 23Tm filaments exhibited calcium-sensitive regulation. All other filaments were inhibitory regardless of whether or not calcium was present.

Effect of Tropomyosin Mutations on Binding to Myosin-free Actin and to Myosin S1-decorated Actin-- To determine whether the inhibitory properties of the tropomyosins were related to destabilization of the myosin-activated conformation of the thin filament, their affinities for actin were determined in the absence and in the presence of myosin S1. Deletion of quasi-repeating regions 2, 3, and 4 has little effect on the affinity of tropomyosin for myosin-free actin (8), suggesting that these regions have little interaction with actin under such conditions. This issue was explored with the new mutants, with representative results shown in Fig. 3A and a data summary presented in the right side of Table II. Both Delta 345Tm and Delta 456Tm were able to displace Delta 234Tm from actin (Fig. 3A), indicating that the affinities of all three tropomyosins were similar. More precisely, the actin affinity of Delta 456Tm (crosses) relative to Delta 234 was 0.75 ± 0.08, and the corresponding affinity ratio for Delta 345Tm (squares) was 0.43 ± 0.07. These data were obtained by competitive displacement assay (30) because Delta 345Tm and Delta 456Tm lack the Cys190 labeling site. Control experiments using unlabeled Delta 234Tm showed it to have a relative affinity compared with labeled Delta 234Tm of 0.9 ± 0.2 (data not shown). In other experiments, Delta 23Tm and Delta 34Tm were labeled on Cys190, and the binding of each to actin was measured directly. Their actin affinities, 1.53 ± 0.03 and 2.02 ± 0.02 × 106 M-1, were each similar that of full-length ASTm, 1.6 × 106 M-1. Table II shows that, in the absence of myosin and of troponin, full-length tropomyosin binds to actin with at most a 4-fold higher affinity than any of the deletion mutants. This strongly suggests that regions 2, 3, 4, 5, and 6 contribute little to actin binding under these conditions. This is consistent with other evidence that it is the NH2 and COOH termini of tropomyosin which are particularly important for this process (20, 39, 48-51).


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  		Fig. 3.   Large and specific effects of internal deletions on tropomyosin binding to actin-myosin S1 and small effects on binding to actin. Panel A, in the absence of myosin, binding of Delta 345Tm (squares and dashed line) or Delta 456Tm (crosses and solid line) to actin was measured by competitive displacement of 3H-labeled Delta 234Tm. Increasing concentrations of unlabeled competing tropomyosin were added to otherwise identical samples, with the results fit to derive the affinities of competing tropomyosins relative to that of Delta 234Tm: 0.75 ± 0.08 for Delta 456Tm and 0.43 ± 0.07 for Delta 345Tm. The affinity of Delta 234Tm for actin under these conditions (60 mM KCl) is 9 × 105 M-1 (not shown), and full-length tropomyosin binds similarly with Kapp = 1.6 × 106 M-1 (8). None of these deletions makes a critical difference in the binding of tropomyosin to actin. Panel B, same as panel A except for the addition of saturating concentrations of myosin S1. The affinity of Delta 345Tm for actin-myosin S1 relative to that of Delta 234Tm was 0.014 ± 0.004. For Delta 456Tm the relative affinity was 0.83 ± 0.06. Under the conditions of the experiment (60 mM KCl), Delta 234Tm binds to actin-myosin S1 with an affinity of 5.3 × 106 M-1 (8), and full-length tropomyosin has an affinity that is too tight to measure even if the KCl concentration is increased to 300 mM. Panel C, binding of radiolabeled Delta 23Tm to actin-myosin S1 was measured directly in the presence of either 150 mM KCl (+; Kapp > 8 × 106 M-1) or 300 mM KCl (×; Kapp = 9.7 ± 0.3 × 105 M-1 and y = 15 ± 2). Panel D, for each of these KCl concentrations weaker binding to actin-myosin S1 was observed for Delta 34Tm. 150 mM KCl (+): Kapp = 1.65 ± 0.04 × 106 M-1 and y = 12 ± 1. 300 mM KCl (×): Kapp < 8 × 104 M-1. Competition assay samples contained 5 µM actin, 3.3 µM 3H-labeled Delta 234Tm, 60 mM KCl, 10 mM NaH2PO4 (pH 7.0), 5 mM MgCl2, and 1 mM EGTA. The data were fit to Equation 1 in Ref. 30, producing the calculated curves shown in panels A and B.

                              
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Table II
Affinities of tropomyosins and of tropomyosin-troponin complexes for F-actin
Control and mutant tropomyosin binding to actin was determined in the presence and absence of troponin. Tropomyosin-troponin data are from Fig. 4 for Delta 23Tm, Delta 34Tm, Delta 345Tm, and Delta 456Tm. Corresponding data for Delta 234Tm and ASTm were obtained similarly, using labeled troponin. Data in the absence of troponin are from Fig. 3A, experiments described in the text, and Ref. 8. Conditions in the presence of troponin were 10 mM NaH2PO4 (pH 7.0), 5 mM MgCl2, 150 mM KCl, 4.5 µM actin, and either 0.5 mM EGTA or 0.1 mM CaCl2. To strengthen tropomyosin-actin binding, the KCl concentration was decreased to 60 mM for the experiments performed in the absence of troponin. The apparent affinity constants (Kapp) include contributions (y) from cooperative tropomyosin-tropomyosin interactions, as well as from actin-binding per se (27, 28). Tm-Tn refers to the tropomyosin-troponin complex. ND means not determined.

In contrast to the above results, the deletions had large and sequence-specific effects on tropomyosin binding to myosin S1-decorated actin. The affinities of Delta 345Tm and Delta 456Tm for actin-myosin S1 were examined by competition with labeled Delta 234Tm (Fig. 3B). Because Delta 234Tm binds to actin-myosin S1 at least 2 orders of magnitude more weakly than does full-length ASTm (8), these experiments required ionic conditions (60 mM KCl) that were favorable for binding. As shown in Fig. 3B, Delta 456Tm (crosses) displaced Delta 234Tm from actin-myosin S1, with a relative affinity of 0.83 ± 0.06. Both tropomyosins bind poorly, but approximately equally. In contrast, Delta 345Tm (squares) was not even able to displace the weakly binding Delta 234Tm, with an affinity of 0.014 ± 0.04 relative to the binding affinity of Delta 234Tm. Poor binding of Delta 345Tm to actin-myosin S1 was also confirmed by SDS-polyacrylamide gel electrophoresis (data not shown).

Full-length ASTm binds to actin-myosin S1 with an affinity too tight to measure reliably by sedimentation with actin; its affinity is at least 107 M-1 even in the presence of 300 mM KCl (8). Under these same high ionic strength conditions both Delta 23Tm (×, Fig. 3C) and Delta 34Tm (×, Fig. 3D) bound much more weakly, particularly Delta 34Tm, with Kapp < 8 × 104 M-1. Decreasing the KCl concentration to 150 mM enhanced the binding of both of these tropomyosins to actin-myosin S1 (+, Fig. 3, C and D). Comparisons between the two panels show that Delta 34Tm, which is inhibitory, bound to actin-myosin S1 an order of magnitude more weakly than did Delta 23Tm, regardless of the KCl concentration.

Effect of the Deletions on Troponin-Tropomyosin Binding to Actin-- To understand the inhibitory properties of the mutants, it was important to determine the mutations' effects on troponin-tropomyosin binding to actin. Two of the mutant tropomyosins lack Cys190, so a novel method for monitoring binding of troponin-tropomyosin to F-actin was applied, using radiolabeled troponin instead of radiolabeled tropomyosin. In support of the method's validity, the stoichiometry of binding depended upon the length of the tropomyosin. Grouping the tropomyosins by length and averaging, saturating amounts of bound troponin-tropomyosin in the presence of 4.5 µM actin were 0.64 ± 0.04 µM for full-length ASTm, 0.93 ± 0.01 µM for tropomyosins spanning five actins, and 1.01 ± 0.02 µM for tropomyosins spanning four actins (data not shown).

Fig. 4, A and B, presents normalized troponin-tropomyosin binding isotherms for the new deletion mutants, and Table II summarizes these results and those for control ASTm and Delta 234Tm. For troponin-Delta 23Tm, troponin-Delta 34Tm, troponin-Delta 345Tm, and troponin-Delta 456Tm, binding to actin occurred cooperatively, with similar affinity, and was weakened modestly by calcium. Affinities in the absence of calcium ranged from a low value of 4.0 × 106 M-1 to a high value 8.3 106 M-1 for Delta 345Tm and Delta 23Tm, respectively. The ratios of the Kapp values in the absence versus the presence of calcium were: 1.61, 1.84, 1.23, 1.23, 1.44, 1.56 for ASTm, Delta 234Tm, Delta 345Tm, Delta 456Tm, Delta 23Tm, and Delta 34Tm, respectively. Although Fig. 4 and Table II show modest differences among the various tropomyosins, the most prominent feature is that none of the deletions had a major effect on troponin-tropomyosin binding to actin, regardless of whether or not Ca2+ was present.


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  		Fig. 4.   Binding of troponin-tropomyosin complexes to actin. Normalized, fractional saturation of actin is shown as a function of increasing concentrations of troponin plus deletion mutant tropomyosin. The conditions are as described under "Materials and Methods," with the KCl concentration 150 mM. Panel A, 0.5 mM EGTA; panel B, 0.1 mM CaCl2. Circles, Delta 345Tm; squares, Delta 456Tm; crosses, Delta 23Tm; triangles, Delta 34Tm. Best fit values for the binding data were determined, were used to generate the curves shown, and are listed in Table II. Binding of troponin-tropomyosin complexes was measured using labeled troponin for Delta 345Tm and Delta 456Tm and with tropomyosins labeled on Cys190 for Delta 23Tm and Delta 34Tm. All of the complexes bound similarly to actin and also bound similarly to control troponin-tropomyosin (Table II), with no more than 3-fold differences in affinity constants. Also, calcium had similar effects on all of the complexes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fig. 5 summarizes the very unfavorable effects of the deletions on the free energy (Delta Delta G) of tropomyosin binding to actin-myosin S1 (dashes), and, by contrast, the small effects on binding in the absence of myosin (circles, squares, diamonds). All of the mutants had impaired binding to actin-myosin S1 (positive Delta Delta G), but the smallest defect was for Delta 23Tm, which is the only form that permitted calcium-sensitive activation of myosin cycling. This suggests that the ability of tropomyosin to bind tightly to actin-myosin is a requirement for activation. However, it is also true that Delta 23Tm is the only mutant that still retains repeat region 4. Therefore, one possibility is that a specific interaction between troponin and repeat region 4 could be the crucial factor distinguishing this tropomyosin from the other, inhibitory forms.


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  		Fig. 5.   Effects of the deletions on the free energy of tropomyosin binding to actin-myosin S1 (bold dashes), to actin (circles), or of troponin-tropomyosin binding to actin in the presence (diamonds) or absence (squares) of calcium. The deletions have small effects unless myosin is present, when defective binding occurs and is dependent upon the specific mutation. Delta Delta G was calculated as -RT ln (fold-change in Kapp) for each mutant relative to full-length recombinant ASTm. Data in the absence of myosin are from Table II. Data in the presence of myosin are from Fig. 3 or from Ref. 8 and incorporate information obtained under more than one ionic condition. For Delta 345Tm and Delta 456Tm, data relative to Delta 234Tm obtained in the presence of 60 mM KCl were combined with data for Delta 234Tm relative to ASTm obtained in the presence of 300 mM KCl (8); the plotted values (bars) for Delta 234Tm, Delta 345Tm, and Delta 456Tm relative to each other depend upon the 60 mM KCl data. For ASTm, Delta 234Tm, Delta 23Tm, and Delta 34Tm data obtained in the presence of 300 mM KCl were used exclusively. Kapp for ASTm binding to actin-myosin S1 was taken as 9 × 106 M-1 under this condition, but binding was nearly stoichiometric so this is a minimum estimate. As a result, the plotted values for the deletion mutants are minimum estimates relative to ASTm, but this consideration does not affect their values relative to each other.

Fig. 5 allows estimation of the relative importance of the repeating regions of tropomyosin on its affinity for myosin S1-decorated actin. There is a notable staircase pattern to the data. The free energy scales with how many of regions 3, 4, and 5 are missing; deletion of one, two, or three of these regions produces serially greater loss of binding. Region 4 appears more significant than region 2 because Delta 34Tm is more defective than is Delta 23Tm. Region 2 appears to contribute little, because Delta 34Tm and Delta 234Tm behave similarly. Region 5 contributes significantly, because Delta 345 is much more defective than is Delta 34 and because Delta 345 is the most impaired of all of the mutants. Region 6 contributes much less than region 3 (Delta 456 versus Delta 345) and apparently no more than does region 2. Regardless of whether some of the details of this additive analysis are faulty because of unknown aspects of the protein structures, the overall pattern is that a broad internal portion of tropomyosin contributes to the molecule's tight binding to myosin S1-decorated actin. This is expected if the repeating tropomyosin alpha -sites are actin-binding motifs that participate in the myosin-induced, active state of the thin filament. In contrast, our data provide evidence against the thesis that these sites are actin binding motifs in the absence of myosin. Under these conditions the deletions produce much smaller effects on the binding free energy.

Hitchcock-DeGregori and colleagues found that deletion of regions 2 or 3 does not interrupt regulation and also that these regions contribute modestly if at all to troponin-tropomyosin binding to actin (34, 35). They further showed that exon 6 (residues 189-213 in repeats 5-6) has little effect on TnT binding to tropomyosin or on troponin-tropomyosin binding to actin. Deletion of half-regions does not affect regulation (34, 52), despite in one case (deletion of residues 191-211; see Ref. 52) removal of part of the alpha -site of tropomyosin repeat 6 (defined as residues 205-224 in Ref. 33). In unpublished work, an unacetylated tropomyosin similar to Delta 234Tm was inhibitory in the presence and in the absence of Ca2+, and one similar to Delta 23Tm allowed partial Ca2+-sensitive activation.3 None of these results conflicts with the current data. However, when internal deletions are made in unacetylated tropomyosin there may be larger effects on troponin-tropomyosin binding to actin than found in the present work.

Comparisons between tropomyosins of different lengths could reflect differences in the strength of end-to-end tropomyosin-tropomyosin contacts. This is because the lengths of the deletions (77 or 119 residues) give primacy to preservation of the heptad repeat and secondary importance to the number of coiled-coil residues (39 1/3) required to span one actin in the filament (33). However, all of the tropomyosin binding curves are cooperative, so this is not likely to be a major factor. It is unclear why there are some differences in the degree of cooperativity, even for tropomyosins of the same length (Table II and Ref. 35).

The present study suggests the importance of a largely unnoticed feature of thin filament behavior: the extremely tight binding of tropomyosin to myosin-decorated actin (10, 53, 54). Tropomyosin in the Ca2+ state sterically blocks part of the myosin binding site on actin (5). Additional, myosin-induced movement of tropomyosin beyond this position cannot easily be explained by steric blocking because myosin S1 produces much tighter (rather than weaker) association of tropomyosin with the thin filament. This greater Kapp could be caused by direct myosin binding to tropomyosin. However, because myosin binding causes spectroscopically detectable changes in F-actin (55-57), it is more likely that myosin allosterically enhances tropomyosin binding via a conformational change in actin. The major regulatory significance of such a conformational change is apparent from the magnitude of the relevant myosin-tropomyosin cooperative interaction. The measured effect of myosin on tropomyosin binding to actin is > 100-fold (10), and deletional analysis implies differences as large as 7,000-fold (Delta 345Tm versus ASTm). These large values are consistent with estimates of the same process that can be made from published data on the energetically linked process of myosin S1 binding to actin. There is a 4-7-fold increase in binding of myosin S1 to actin-tropomyosin or actin-tropomyosin-troponin-Ca2+ when either is compared with binding to actin alone (12, 58, 59). Equilibrium linkage calculation using the most conservative of these data (58) translates to an 8,000-fold effect of myosin S1 on the binding of the regulatory proteins to actin ((Kstrong/Kunregulated actin)7/L' = 47/2 = 8,000) (18).

We suggest that myosin binding causes a conformational change in actin, producing a distinct, highly favorable site for tropomyosin binding. The deletion mutants weaken this movement and conformational change, perhaps via binding of fewer tropomyosin alpha -sites to actin-myosin, particularly sites 3, 4, and 5 that are predominantly responsible for the effect. In this proposal tropomyosin has both steric and allosteric roles. It sterically blocks myosin binding (to a greater or lesser extent depending upon Ca2+ binding to troponin) but allosterically promotes an actin conformational change that occurs when myosin binds to actin. Myosin binding promotes tropomyosin movement, and repositioned tropomyosin enhances myosin binding by promoting the actin conformational change.

Although the above mechanism is sufficient to explain the energetics of the azimuthal movement of tropomyosin in the presence of myosin, the mechanism lacks kinetic detail. One possibility is that when troponin-tropomyosin is in the calcium position (but not when in the EGTA position), enough of the myosin binding site on actin is exposed so that myosin binds and initiates a change in the actin, which in turn produces the additional regulatory protein movement. The structural features of this initial myosin binding cannot be discerned from current atomic models of myosin and of the thin filament. The other possibility is that the final azimuthal change in tropomyosin position must occur first, that only subsequent to this movement does the myosin bind stereospecifically to actin, and that this myosin binding causes actin conformation to change sufficiently so that tropomyosin then interacts strongly in this final position.

The inhibitory tropomyosins of the present report have greatly decreased affinities for actin specifically under one condition, when myosin is bound. This common property suggests that the normal, tight tropomyosin binding to the fully activated (so-called open) state of the thin filament is required for myosin cycling. In this sense the proposed conformational change in actin appears to be an essential part of regulation; it critically facilitates the final troponin-tropomyosin movement away from the myosin binding site on actin, beyond the calcium position. To understand these phenomena further, it may prove useful to explore the effects of the tropomyosin deletions on muscle cross-bridge function and on myosin-actin binding equilibria and kinetics.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants NHLBI-38834 (to L. S. T.) and AR-30988 (to E. H.).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.

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

2 M. Rosol, W. Lehman, R. Craig, C. Landis, and L. S. Tobacman, submitted for publication.

3 S. Hitchcock-DeGregori, personal communication.

    ABBREVIATIONS

The abbreviations used are: myosin S1, myosin subfragment 1; Delta 23Tm, rat Ala-Ser alpha -tropomyosinDelta (47-123); Delta 234Tm, rat Ala-Ser alpha -tropomyosinDelta (47-165); Delta 34Tm, rat Ala-Ser alpha -tropomyosinDelta (80-165); Delta 345Tm, rat Ala-Ser alpha -tropomyosinDelta (89-207): Delta 456Tm, rat Ala-Ser alpha -tropomyosinDelta (124-242); ASTm, rat Ala-Ser alpha -tropomyosin. Delta 23Tm, Gln47 through Ser123; Delta 34Tm, Asn89 through Val165; Delta 345Tm, Asn89 through Leu207; Delta 456Tm, Glu124 through Ala242; TnT, troponin T; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Parry, D. A. D., and Squire, J. M. (1973) J. Mol. Biol. 75, 33-55[CrossRef][Medline] [Order article via Infotrieve]
2. Huxley, H. E. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 361-376
3. Haselgrove, J. C. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 341-352
4. Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771[Abstract/Free Full Text]
5. Vibert, P., Craig, R., and Lehman, W. (1997) J. Mol. Biol. 266, 8-14[CrossRef][Medline] [Order article via Infotrieve]
6. Holmes, K. C. (1995) Biophys. J. 68, 2S-7S
7. Lorenz, M., Poole, K. J. V., Popp, D., Rosenbaum, G., and Holmes, K. C. (1995) J. Mol. Biol. 246, 108-119[CrossRef][Medline] [Order article via Infotrieve]
8. Landis, C. A., Bobkova, A., Homsher, E., and Tobacman, L. S. (1997) J. Biol. Chem. 272, 14051-14056[Abstract/Free Full Text]
9. McKillop, D. F. A., and Geeves, M. A. (1993) Biophys. J. 65, 693-701[Abstract/Free Full Text]
10. Cassell, M., and Tobacman, L. S. (1996) J. Biol. Chem. 271, 12867-12872[Abstract/Free Full Text]
11. Williams, D. L., Greene, L. E., and Eisenberg, E. (1988) Biochemistry 27, 6987-6993[CrossRef][Medline] [Order article via Infotrieve]
12. Trybus, K. M., and Taylor, E. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7209-7213[Abstract/Free Full Text]
13. Guth, K., and Potter, J. D. (1987) J. Biol. Chem. 262, 13627-13635[Abstract/Free Full Text]
14. Butters, C. A., Tobacman, J. B., and Tobacman, L. S. (1997) J. Biol. Chem. 272, 13196-13202[Abstract/Free Full Text]
15. Bremel, R. D., Murray, J. M., and Weber, A. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 267-275
16. Cantino, M. E., Allen, T. S., and Gordon, A. M. (1993) Biophys. J. 64, 211-222[Abstract/Free Full Text]
17. Ishii, Y., and Lehrer, S. S. (1990) Biochemistry 29, 1160-1166[CrossRef][Medline] [Order article via Infotrieve]
18. Hill, T. L., Eisenberg, E., and Greene, L. E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3186-3190[Abstract/Free Full Text]
19. Ruiz-Opazo, N., and Nadal-Ginard, B. (1987) J. Biol. Chem. 262, 4755-4765[Abstract/Free Full Text]
20. Willadsen, K. A., Butters, C. A., Hill, L. E., and Tobacman, L. S. (1992) J. Biol. Chem. 267, 23746-23752[Abstract/Free Full Text]
21. Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871[Abstract/Free Full Text]
22. Weeds, A. G., and Taylor, R. S. (1975) Nature 257, 54-56[CrossRef][Medline] [Order article via Infotrieve]
23. Tobacman, L. S., and Adelstein, R. S. (1986) Biochemistry 25, 798-802[CrossRef][Medline] [Order article via Infotrieve]
24. Homsher, E., Wang, F., and Sellers, J. R. (1992) Am. J. Physiol. 262, C714-C723[Abstract/Free Full Text]
25. Tobacman, L. S., and Lee, R. (1987) J. Biol. Chem. 262, 4059-4064[Abstract/Free Full Text]
26. Mehegan, J. P., and Tobacman, L. S. (1991) J. Biol. Chem. 266, 966-972[Abstract/Free Full Text]
27. Hill, L. E., Mehegan, J. P., Butters, C. A., and Tobacman, L. S. (1992) J. Biol. Chem. 267, 16106-16113[Abstract/Free Full Text]
28. McGhee, J. D., and von Hippel, P. H. (1974) J. Mol. Biol. 86, 469-489[CrossRef][Medline] [Order article via Infotrieve]
29. Tsuchiya, T., and Szabo, A. (1982) Biopolymers 21, 979-994[CrossRef][Medline] [Order article via Infotrieve]
30. Hinkle, A., Goranson, A., Butters, C. A., and Tobacman, L. S. (1999) J. Biol. Chem. 274, 7157-7164[Abstract/Free Full Text]; correction (1999) J. Biol. Chem. 274, 31750
31. Homsher, E., Kim, B., Bobkova, A., and Tobacman, L. S. (1996) Biophys. J. 70, 1881-1892[Abstract/Free Full Text]
32. Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4682-4690[Abstract/Free Full Text]
33. McLachlan, A. D., and Stewart, M. (1976) J. Mol. Biol. 103, 271-298[CrossRef][Medline] [Order article via Infotrieve]
34. Hitchcock-DeGregori, S. E., and Varnell, T. A. (1990) J. Mol. Biol. 214, 885-896[CrossRef][Medline] [Order article via Infotrieve]
35. Hitchcock-DeGregori, S. E., and An, Y. (1996) J. Biol. Chem. 271, 3600-3603[Abstract/Free Full Text]
36. Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466[Abstract/Free Full Text]
37. Meedel, T. H., and Hastings, K. E. M. (1993) J. Biol. Chem. 268, 6755-6764[Abstract/Free Full Text]
38. Ishii, Y., and Lehrer, S. S. (1991) J. Biol. Chem. 266, 6894-6903[Abstract/Free Full Text]
39. Hammell, R. L., and Hitchcock-DeGregori, S. E. (1996) J. Biol. Chem. 271, 4236-4242[Abstract/Free Full Text]
40. Phillips, J. G. N., Lattman, E. E., Cummins, P., Lee, K. Y., and Cohen, C. (1979) Nature 278, 413-417[CrossRef][Medline] [Order article via Infotrieve]
41. Fisher, D., Wang, G., and Tobacman, L. S. (1995) J. Biol. Chem. 270, 25455-25460[Abstract/Free Full Text]
42. Butters, C. A., Willadsen, K. A., and Tobacman, L. S. (1993) J. Biol. Chem. 268, 15565-15570[Abstract/Free Full Text]
43. Morris, E. P., and Lehrer, S. S. (1984) Biochemistry 23, 2214-2220[CrossRef][Medline] [Order article via Infotrieve]
44. Dahiya, R., Butters, C. A., and Tobacman, L. S. (1994) J. Biol. Chem. 269, 29457-29461[Abstract/Free Full Text]
45. Tanokura, M., and Ohtsuki, I. (1984) J. Biochem. 95, 1417-1421[Abstract/Free Full Text]
46. Mak, A. S., and Smillie, L. B. (1981) J. Mol. Biol. 149, 541-550[CrossRef][Medline] [Order article via Infotrieve]
47. Cabral-Lilly, D., Tobacman, L. S., Mehegan, J. P., and Cohen, C. (1996) Biophys. J. 73, 1763-1770[Abstract/Free Full Text]
48. Babu, A., Su, H., Ryu, Y., and Gulati, J. (1992) J. Biol. Chem. 267, 15469-15474[Abstract/Free Full Text]
49. Heald, R. W., and Hitchcock-DeGregori, S. E. (1988) J. Biol. Chem. 263, 5254-5259[Abstract/Free Full Text]
50. Cho, Y.-J., Liu, J., and Hitchcock-DeGregori, S. E. (1990) J. Biol. Chem. 265, 538-545[Abstract/Free Full Text]
51. Mabuchi, K. (1996) J. Structural Biol. 116, 278-289[CrossRef][Medline] [Order article via Infotrieve]
52. Hammell, R. L., and Hitchcock-DeGregori, S. E. (1997) J. Biol. Chem. 272, 22409-22416[Abstract/Free Full Text]
53. Eaton, B. L. (1976) Science 192, 1337-1339[Abstract/Free Full Text]
54. Cregg, J. M., Vedvick, T. S., and Raschke, W. C. (1993) Bio/Technology 11, 905-910[CrossRef][Medline] [Order article via Infotrieve]
55. Feng, L., Kim, E., Lee, W.-L., Miller, C. J., Kuang, B., Reisler, E., and Rubenstein, P. (1997) J. Biol. Chem. 272, 16829-16837[Abstract/Free Full Text]
56. Kouyama, T., and Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38[Medline] [Order article via Infotrieve]
57. Criddle, A. H., Geeves, M. A., and Jeffries, T. (1985) Biochem. J. 232, 343-349[Medline] [Order article via Infotrieve]
58. Williams, D. L., and Greene, L. E. (1983) Biochemistry 22, 2770-2774[CrossRef][Medline] [Order article via Infotrieve]
59. Geeves, M. A., and Halsall, D. J. (1986) Proc. R. Soc. Lond. B Biol. Sci. 229, 85-95[Medline] [Order article via Infotrieve]

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