Analysis of Troponin-Tropomyosin Binding to Actin

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 23, Issue of August 15, pp. 16106-16113,1992 Printed in U. S. A.

Analysis of Troponin-Tropomyosin Binding to Actin TROPONIN DOES NOT PROMOTE INTERACTIONS BETWEEN TROPOMYOSIN MOLECULES*

(Received for publication, February 18, 1992)

Laura E. Hill, John P. Mehegan, Carol A. Butters, and Larry S . TobacmanS From the Departments of Internal Medicine and Biochemistry, College of Medicine, University of Zowa, Iowa City, Iowa 52242

The binding of tropomyosin to actin and troponin- tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: KO, the affinity of the ligand for an isolated site on the actin filament, and y, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative ( y = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in KO, while cooperativity ac- tually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 ( t r o p ~ n i n , ~ - ~ ~ ~ ) or 1-158 ( t r o p ~ n i n ~ ~ ~ - ~ ~ ~ ) were examined. Deletion of amino acids 1-69 had only small effects on KO and y, despite this peptide’s location spanning the joint between adjacent tropomyosins. Ca2+ reduced KO by half for both troponin and troponin,^-^^^ and had no detectable effect on cooperativity. had much weaker effects on tropomyosin-actin binding than did t r o p ~ n i n ~ ~ - ~ ~ ~ and had no effect at all in the presence of Ca2+. This suggests the importance of CaZ+-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for t r 0 p o n i n ~ ~ 9 - ~ ~ ~ than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.

Skeletal muscle contraction is regulated by the reversible binding of Ca2+ to the thin filament protein troponin. Among the approaches used to understand this regulatory process have been the identification and measurement of specific interactions among the five polypeptides comprising the thin filament: actin, tropomyosin, and the three troponin subunits TnI, TnC, and TnT. Some of these interactions are sensitive to Ca2+, and some are perturbed by actin-myosin binding (Ingraham and Swenson, 1984; Williams et al., 1988; Potter and Gergely, 1974; Mak et al., 1983). Based in part upon these results, several useful models for the molecular basis of regulation have been proposed (e.g. Huxley, 1972; Hill, 1983; Phillips et al., 1986; Heeley et al., 1987). These models have been constrained by the absence of detailed structural infor- mation for the assembled thin filament. However, recent progress determining the structures of both actin (Holmes et al., 1990; Milligan, 1990) and troponin C (Herzberg and

* This investigation was supported by National Institutes of Health Grant HL38834 and by a Grant-in-Aid from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a CIBA-Geigy Established Investigatorship from the American Heart Association.

James, 1988) will encourage further investigation of these models.

In some respects, the complexity and size of the thin filament hamper attempts to discern and measure specific interactions within it. On the other hand, these features of the actin filament can be turned to advantage, as the present study intends. Tropomyosin binding to actin involves a parking problem in which energetic considerations must overcome the statistical difficulty of positioning neighboring tropomyosins end-to-end without gaps between them. Energetically favorable interactions between adjacent tropomyosin molecules are an essential part of thin filament assembly. Fur- thermore, these interactions can be measured by an equilibrium analysis of tropomyosin-actin binding, using the math- ematical formulation for binding of a ligand to a linear lattice (McGhee and von Hippel, 1974). In principle, an identical analysis can be performed for troponin-tropomyosin binding to actin. In this paper we identify conditions where tropomyosin binding to actin and troponin-tropomyosin binding to actin can be compared. Surprisingly, we find that troponin decreases rather than increases the strength of interactions between thin filament-bound tropomyosin molecules. We also describe the effect of Ca2+ on thin filament assembly, as well as the effects of deleting TnT residues 1-69 (the tropomyosin- tropomyosin overlap peptide) and TnT residues 1-158 (the 15 nm long TnT tail) on troponin-tropomyosin binding to actin.

MATERIALS AND METHODS

Preparation of Proteins-Rabbit fast skeletal muscle actin was prepared by the method of Spudich and Watt (1971). Rabbit fast skeletal muscle troponin and troponin subunits were prepared according to Potter (1982), omitting the Cibacron Blue step. Tropo- myosin was isolated from the bovine heart as previously reported (Tobacman and Adelstein, 1986).

Troponin containing TnI, TnC, and T n T residues 159-259 was prepared by lightly digesting whole troponin with chymotrypsin, using the procedure of Morris and Lehrer (1984). After digesting 1.4 mg/ ml troponin at 1:750 weight ratio (chymotrypsin/troponin) for 45 min at 4 “C in the presence of 10 mM imidazole (pH 7.5), 0.5 mM dithiothreitol, and 0.01% NaN3, the reaction was quenched with phenylmethylsulfonyl fluoride and Ne-p-tosyl-L-lysine chloromethyl ketone. The truncated troponin complex was isolated by chromatography over Q Sepharose and its subunit composition was confirmed by SDS- PAGE.’ The identity of the TnT fragment was further established by NH2-terminal sequencing after SDS-PAGE and electrophoretic transfer to a Problot membrane. Six cycles of Edman degradation on an Applied Biosystems Sequencer demonstrated the sequence Leu- Ala-Lys-Ala-Asp-Gln, matching TnT residues 159-164. The complex was designated troponinl59-259.

Isolation of Recombinant Rat Skeletal Muscle TnT Residues 70-

’ The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; dibromo BAPTA, [1,2-bis-(2- amino-5-bromo-phenoxy)ethane-~,N,N’,N’tetraacetic acidltetraso- dium salt.

16106

Troponin-Tropomyosin Binding to Actin 16107

259"To isolate a fragment of T n T lacking the peptide that spans the tropomyosin-tropomyosin overlap joint, we engineered a prokar- yotic expression vector from rat skeletal muscle T n T cDNA provided by Drs. Breitbart and Nadal-Ginard (Harvard Medical School) (Breit- bart et al., 1985). The pBR325 plasmid pTnTIII was restricted with PstI by standard methods (Maniatis et al., 1982; Davis et al., 19861, and the TnT cDNA insert was ligated into pSP72. The DNA sequence was confirmed by dideoxynucleotide sequencing (Sanger et al., 1977) from both directions. The TnT (70-259) DNA was obtained by restricting the resultant plasmid at an internal AuaII site, blunting with mung bean nuclease, cutting with BamHI, and isolating the excised fragment from an agarose gel. The 5' end of this fragment is 1 base pair upstream from the codon for Met-70, which we used as a translation start site. The fragment was inserted into pET3d, a gift from Dr. William Studier (Brookhaven) (Studier et al., 1990), which bad been opened with NcoI, treated with mung bean nuclease, and then restricted with BamHI. Proper insertion was confirmed by dideoxynucleotide sequencing, using the T7 promoter of pET3d as the primer site. The expression plasmid, designated pTnT70, was transfected into three cell lines carrying the gene for T7 polymerase: DE3, DE3 Lys S , and DE3 Lys E. Optimal T n T (70-259) expression was obtained with the DE3 cells grown overnight in the absence of isopropyl-1-thio=P-ldz-galactopyranoside. The reason for this is unclear, but TnT may be toxic to DE3 cells. The cells were homogenized and frozen in an extraction buffer containing 6 M urea, 1 M KCI, and 10 mM Tris HCI (pH 7.0). After low speed centrifugation, the soluble extract was dialyzed overnight against 2 M urea, 10 mM Tris (pH 7.5), and 0.3 mM phenylmethylsulfonyl fluoride. The TnT (70-259) precipitated in this low ionic strength buffer. It was further purified with a hydroxylapatite column, from which it was eluted with a 0- 200 mM K,HP04 gradient. The protein was shown to be homogeneous by SDS-PAGE, and its identity was confirmed by NHz-terminal amino acid sequencing. Typical yields were 10-20 mg of T n T (70- 259)lliter of bacterial culture. T n T (70-259) was reconstituted with rabbit fast skeletal muscle TnI and TnC as described by Potter (19823, with a final Sephadex G-100 chromatography step included to separate the troponin complex from free subunits. This complex was designated troponin70-zsy.

Measurement of Tropomyosin Binding to Actin-To sensitively determine tropomyosin binding to F-actin, tropomyosin was labeled on Cys-39 with i~do[~H]acetic acid (Amersham Corp.), following Hitchcock-DeGregori and Varnell's method (1990) except that the pH of the tropomyosin solution was raised to 7.5 before the label was added. Incorporation was 1.8-2.2 mol/mol. The resultant tropomyosin specific activity was 3.3 X 10' cpm/pmol. Tropomyosin binding to actin was measured by sedimentation of samples in a Beckman Airfuge at 30 psi for 20 min. The radioactivity in duplicate 15-pI aliquots was measured before and after centrifugation to determine free and bound tropomyosin concentrations. In preliminary experiments the radioactivity in the supernatants was poorly reproducible. Control samples with low tropomyosin concentrations and no other proteins lost radioactivity on simple transfer into and out of centri- fuge tubes. Reproducible and coherent results were obtained hy including 0.3 mg/ml bovine serum albumin in all samples to eliminate nonspecific surface losses. A control experiment using silonized materials and no albumin was conducted after most of the results in this paper were completed. It produced data indistinguishable from the results with albumin (data not shown).

Depending upon the protein concentration and the age (up to 1 month) of the F-actin preparation, between 70 and 95% of total actin pelleted according to protein assay measurements (Bradford, 1976) of samples without troponin or tropomyosin and according to SDS- PAGE of samples including troponin-tropomyosin. Other experiments implied that any non-pelleted actin was not polymerized under conditions where tight tropomyosin-actin binding was expected (excess concentrations of actin and troponin), more than 97% of the tropomyosin sedimented (data not shown). Except as indicated, binding of troponin-tropomyosin to actin was measured a t 23 "C in the presence of 5 p M polymerized actin, 60 mm or 300 mM KC1, 3 mM MgCL, 0.3 mg/ml bovine serum albumin, 0.5 mM dithiothreitol, 0.5 mM dibromo BAPTA, and 10 mM Tris-HCI (pH 7.0).

The free Ca+* concentration was controlled by including either 0.5 mM dibromo BAPTA or 0.5 mM dibromo BAPTA plus 0.6 mM CaCIZ in experimental samples, producing estimated Caf2 concentrations of lo-' M or 10" M, respectively (Tohacman, 1987; Tobacman and Sawyer, 1990).

To assess any possible effect of the labeling procedure, a series of samples was studied containing 10 g M F-actin and 2 p~ total tropo-

myosin consisting of mixtures of labeled and unlabeled molecules (k0, 1:1, 1:3, and 1:7 ratios of labeled/unlabeled). The proportion of labeled tropomyosin binding to actin (40%) was unaffected by the dilution with unlabeled molecules, suggesting that the labeling procedure did not influence binding.

To measure the binding of the troponin-tropomyosin complex to F-actin, troponin was added in 0.4 p M excess over tropomyosin. Still higher concentrations of troponin do not measurably increase tropomyosin-actin binding (Mehegan and Tobacman, 1991; data not shown). This is qualitatively consistent with tropomyosin binding to troponin with a high affinity of 6.7 X lo6 M" (Morris and Lehrer, 1984). Labeling tropomyosin with dansylaziridine (Ingraham and Swenson, 1985) or pyrene (Ishii and Lehrer, 1991) increases troponin affinity. The troponin affinity of carboxymethylated tropomyosin has not been measured, but we found 0.4 p M free troponin to saturate the effect of troponin in all experiments. The one exception, as indicated below, is that higher concentrations were required for troponin1s9.25y. For the purposes of calculation, all of the measured free tropomyosin and all of the measured bound tropomyosin were assumed to be troponin-tropomyosin complexes. Any error in this assumption would affect the free troponin-tropomyosin concentration more than the actin-hound troponin-tropomyosin concentration, since troponin promotes tropomyosin-actin binding and detailed balance implies that actin must promote troponin-tropomyosin binding. On the other hand, the above binding constant suggests that 0.4 p M excess troponin might have failed to saturate 25% of the non-actin-bound tropomyosin molecules resulting in a overestimate of the free troponin- tropomyosin concentration. Computer simulation suggests this would result in a 25% underestimate for the value of K,, reported in this paper, but would have no effect on y (see below).

Analysis of Binding Curues-Parameter estimation was performed hy non-linear least squares analysis using two commercially available personal computer programs, MATLAB (Mathworks Inc.) and MINSQ (MicroMath Scientific Software). For a ligand binding to every seventh element of a linear lattice, the bound ligand ( 0 ) is an implicit function of the free ligand ( L ) (Zasedatelev, et al., 1971; McGhee and von Hippel, 1974; Hill, 1985)

L = 4u(l - u/N)'[2(y - 1)(1 - u/N)I6

7K0N(1 - 8u/N/7 + R)'[(2y - 1)(1 - u/N) + u/N/7 - RI6(1 - u / N ) (1)

R = [ ( l - 8~/N/7) ' + 4y~(l - U/N)/N/~]" '

where KO is the affinity of the ligand (tropomyosin or troponin- tropomyosin) for an isolated site on the lattice, yK, is the affinity of the ligand for the lattice when there is one adjacent, already-bound ligand, and N is the maximum concentration of bound ligand (the F- actin concentration divided by seven). In other words, KO is an affinity constant, y is a cooperativity parameter, and N is proportional to the lattice concentration. (The cooperativity term "Y" in Hill et al., 1980, 1983 is a product of several parameters. One of these is y in this report.) To find the best fit for all three parameters, the MATLAB fmin command was used to numerically solve Equation 1 and obtain the theoretical bound ligand concentration for each experimental free ligand concentration, and the fmins command was used to adjust KO, y, and N to find the set of values producing the least squares error for the entire data set. When provided with the value of N determined by MATLAB, the MINSQ program would converge on the same best fit values of KO and y that were identified by MATLAB, and, in addition, calculate standard errors for these two parameters. MINSQ was also used for initial estimates of y and KO.

The theoretical model is very general in some respects. The protein- protein interactions responsible for the cooperativity can be within the actin lattice itself, as well as between neighboring ligands. How- ever, the model does depend upon several assumptions. No interactions occur across empty stretches of lattice or between ligands that are not adjoining. Furthermore, the model omits possible effects of ligands across the width of the two-start actin filament, considering only longitudinal effects. Finally, ligands bind with only one polarity and never bind closer than seven actins apart.

RESULTS

Binding of Tropomyosin to Actin in the Absence of Tro- ponin-Fig. 1 shows representative data and theoretical curves illustrating the assembly of tropomyosin onto the actin filament in the presence of two different ionic strengths. The

16108 Troponin-Tropomyosin Binding to Actin

A 0.60 , 1

0 00

T r o p o m y o s i n , uM

\ 0.40 t 0.00 1

0.00 0.10 0.20 0.30 0.40 0.50 0.60

Bound Tropomyosin, uM

3.00 t / -

'E 7.50 1 0

c l L

,/

/

~

1 I

0.0 4 0 8 0 1 7 0 1 6 0 2 0 0 2 3 0

Tropomyos in , uM FIG. 1. Binding of tropomyosin to actin. Panel A, binding of

radiolabeled tropomyosin to actin in the presence of 60 mM KC1 and M Ca'+ was assessed by Airfuge centrifugation of samples con-

taining various free tropomyosin concentrations. The abscissa indicates the concentration of free tropomyosin, and the ordinate indicates the concentration of bound tropomyosin. The S-shaped line represents the best fit of the data to the linear-lattice model. It demonstrates a value of binding to an isolated site of KO = 12 2 1.0 X 10" M" and a cooperativity parameter of y = 84 2 7.0. For comparison, two less cooperative curves with the same value for the product, yK,, have been included. The middle curue shows the binding expected if there were less cooperativity (KO = 120 X lo3 M-', y = 8.4). The other curve is calculated assuming negligible cooperativity (K,, = 1000 X loy M" and y = 1.05). Panel B, the data from panel A have been plotted for Scatchard analysis. The conuen line represents the best fit for the data. The other lines show plots with the same yK,, value when y = 1 (no cooperativity), y = 4, and y = 8.4. Panel C, the binding of tropomyosin to actin was measured as in panel A

binding curves passing through the data points in Fig. 1, A and C, are S-shaped, implying a cooperative process. However, because of the statistical difficulty in properly "parking" the tropomyosins along the actin filament, the relationship between the shape of such curves and the presence of cooperativity is complex. Non-cooperative curves appear to be negatively cooperative. S-shaped curves appear only when the level of cooperativity is very high (McGhee and von Hippel, 1974). Half-saturation of binding occurs, to a first approxi- mation, when the free tropomyosin concentration equals 1/ ( yK,). The best fit theoretical curve in panel A has a cooperativity parameter of y = 84. Panel B shows the same data transformed to the axes used for Scatchard analysis. To illustrate the effect of y on the binding curve, additional theoretical curves not fitting the data are shown in panels A and B. All of these curves have the same values for N and for the product yK,, but differing values for the individual parameters y and KO. If the value of y were close to 1, and there were neither positive nor negative cooperativity, the binding pattern would appear to be negatively cooperative (concave on the Scatchard plot). If y were 4, no cooperativity would be apparent on the Scatchard plot (i.e. it becomes almost linear), despite a true, 4-fold cooperative effect.

Table I shows the average values for KO, y, and yK,, from several experiments. Comparison of Fig. 1, panels A and C, and examination of the values in Table I shows that binding is much weaker in the presence of 300 mM KC1 (Fig. IC) than in the presence of 60 mM KC1 (Fig. 1A). Much higher concentrations of tropomyosin were required to measure binding under the higher ionic strength conditions. In the presence of 60 mM KCl, yKo is 17-fold greater than in the presence of 300 mM KCI. This difference is entirely due to altered affinity for an isolated site on the actin filament (KO), which is only 600- 700 M" in the presence of 300 mM KCl, but is 27 times this

TABLE I Binding of tropomyosin or troponin-tropomyosin to actin

Thin filament assembly was analyzed according to Equation 1, under conditions as described under "Materials and Methods." Except as indicated, values are averages of results from two preparations for each condition. Tm represents tropomyosin, troponin is rabbit fast skeletal muscle troponin, reconstituted troponin is rabbit fast skeletal muscle troponin reconstituted from purified subunits, troponin 70- 259 is troponin reconstituted from rabbit TnI and TnC and rat TnT residues 70-259, and troponin 159-259 is rabbit troponin after pro- teolytic removal of T n T residues 1-158 from the troponin complex. KO is the affinity constant of ligand for an isolated site on the actin filament. y is the fold increase in affinity when ligand binding occurs adjacent to one already bound ligand. yK, is the overall affinity constant, per ligand, taking into account isolated site binding as well as cooperative interactions.

KC1 K" Y Y R

pCa8 pCa4 pCa8 pCa4 pCa8 pCa4 10-3 x M' 10-6 x M'

300 mM Tm alone 0.58 0.69 138 136 0.080 0.094 Tm-troponin 85 46 36 39 3.0 1.8 Tm-troponin 70-259 36 15 82 105 3.0 1.6 Tm-reconstituted 47 28 66 52 3.1 1.5

troponin 60 mM

Tm alone" 17 90 1.5 Tm-troponin 159-259 82 34 2.8 a Average of six determinations performed over Ca'+ concentrations

between lo-' M and M.

except [KCI] = 300 mM, (actin] = 21 p ~ , and no calcium was added (estimated pCa 8). The line shows the best fit to Equation 1. K,, = 0.64 & 0.14 X 10" M-', y = 135 2 31.


value under the lower ionic strength conditions. Table I also shows that ionic strength has only a small

effect on y , which is a direct measure of the strength of the interactions between bound tropomyosin molecules. In the presence of 300 mM KC1, y is 137 (experimental range 82 to 190), while the value of y at lower ionic strength was 90 (experimental range 53 to 136). The small effect of KC1 concentration on y was surprising because tropomyosin polymerization is strongly inhibited by raising the ionic strength (Heeley et al., 1988, Tobacman and Lee, 1986).

As discussed below, Fig. 1 is in many respects consistent with data from prior investigations, but previous studies have only rarely been analyzed according to Equation 1 (Wegner, 1979; Wegner and Walsh, 1981). The curve fitting shows that tropomyosin-actin binding is extremely cooperative. The physical interpretation for these values of y is that tropomyosin-actin binding affinity is increased by two orders of magnitude when one neighboring tropomyosin molecule is already on the actin filament. The results further imply how tightly tropomyosin binds to a seven-actin-long gap between two already bound tropomyosins: y2Ko = lo7 M" or 10' M-', respectively, in the presence of 300 or 60 mM KC1. These affinities are approximately four orders of magnitude higher ( y2 = lo4, approximately) than those for binding to an isolated site.

Troponin-Tropomyosin Binding to Actin-The troponin- tropomyosin complex binds very tightly to actin, making this assembly process difficult to analyze quantitatively. To identify conditions suitable for such an analysis, we measured tropomyosin sedimentation alone, with actin, with troponin, and with both troponin and actin, as a function of ionic strength (Fig. 2). In the absence of other proteins, tropomyosin did not precipitate regardless of ionic strength (squares). When actin was added (circles), tropomyosin bound to the F- actin and co-sedimented with it in the presence of low ionic strength. Increasing KC1 concentrations diminished tropomyosin-actin binding. These results are consistent with Fig. 1. Fig. 2 also shows that addition of both troponin and actin (triangles) caused substantially more tropomyosin to pellet than when troponin was omitted, but this did not all represent binding to actin: troponin caused some tropomyosin to pellet even in the absence of actin (diamonds), perhaps by inducing polymerization. This problem would experimentally and con-

' * I -7

'A

- 0 2 I i 0 100 200 300 400

KCI. mM

FIG. 2. Effect of [KCl], actin, and troponin on tropomyosin sedimentation. Tropomyosin sedimentation in varied [KCI] was assessed by Airfuge centrifugation of 0.9 pM tropomyosin alone (squares), with 5 pM F-actin (circles), with 1.4 pM troponin (diamonds) , and with both actin and troponin (triangles). Only in the presence of at least 300 mM KC1 does sedimentation of troponin- tropomyosin imply actin binding.

ceptually confound attempts to measure troponin-tropomyosin binding to actin by sedimentation assay, except for the more favorable results at high ionic strength. In the presence of 300-350 mM KC1, tropomyosin did not sediment when troponin, but not actin, was added. In other experiments performed in the absence of actin, the troponin-tropomyosin concentration was varied in the presence of 300 mM KC1, and the complex was shown not to sediment at concentrations up to 2 FM tropomyosin (data not shown).

Using the conditions identified in Fig. 2 (300 mM KCl), the assembly of the troponin-tropomyosin complex onto the actin filament lattice was investigated. Fig. 3 shows one representative data set for the troponin-induced tropomyosin-actin binding; Table I shows the average values for y , KO, and yKo (two preparations each at pCa 4 and pCa 8). Binding to actin was much tighter when troponin was present, with the overall binding constant yK,, increasing approximately 30-fold, compared to the results with tropomyosin alone (Table I). This higher affinity was entirely due to tighter binding to an isolated site (KO) . Addition of troponin increased KO by two orders of magnitude. There was a small effect of Ca2+ on this process in the direction predicted by other types of experiments. Ca2+ weakened KO by 50%.

Troponin spans the tropomyosin-tropomyosin overlap joint (White et al., 1987) and promotes tropomyosin polymerization in the absence of actin. Therefore, we anticipated that troponin would increase the strength of the interactions between bound tropomyosin molecules and, thus, cooperativity. In- stead, the results showed that troponin had a small effect in the opposite direction. The value of y was decreased by the addition of troponin, falling to an average of 37 (experimental range 24 to 48), compared to a value of 135 (experimental range 82-190) in the absence of troponin. The best fit curve calculated and shown for the troponin-tropomyosin data set

/ B -1

Tropon in -Tropom\ -os l I l . ull FIG. 3. Binding of troponin-tropomyosin to actin in the

presence of 300 mM KCl. Binding of troponin-tropomyosin to actin at high ionic strength was assessed by Airfuge centrifugation of samples containing various tropomyosin and troponin concentrations. Troponin was always present at an apparently saturating molarity, 0.4 p M higher than the concentration of total tropomyosin. The abscissa indicates the concentration of free troponin-tropomyosin, determined from the labeled tropomyosin in the supernatant. Similarly, the bound troponin-tropomyosin concentration was equated to the measured concentration of sedimented tropomyosin. The line through the points is the best fit with KO = 82 f 10 X 10:' M" and y = 49 f 6.0. To demonstrate the difference between troponin-tropomyosin and tropomyosin alone binding to actin, the second line which does not match the points was included. This shows the same value for yKo as troponin-tropomyosin-actin binding, but the value of y is 139, the cooperativity parameter for tropomyosin- actin binding in the absence of troponin.


in Fig. 3 suggests y = 48. The figure also shows the poorer fitting theoretical curve that would result if the value of y were the same as when troponin was omitted ( y = 138).

As indicated in Table I, a t pCa 8, y was 36 (experimental range 24-48) and at pCa 4, y was 39 (experimental range 32- 46). Thus, Ca2+ had no detectable effect on y, although with the large ranges, a small effect could be masked. Prior publi- cations show conflicting data regarding whether Ca2+ affects cooperativity of troponin-unacetlyated tropomyosin binding to actin (Heald and Hitchcock-DeGregori, 1988; Cho et al., 1990; Hitchcock-DeGregori and Varnell, 1990).

Effect of Deletion of TnT Residues 1-69 on Thin Filament Assembly-To further explore the importance of the troponin- tropomyosin polymer on thin filament properties, the region of TnT that spans the tropomyosin-tropomyosin overlap joint (1-69) was deleted from a recombinant T n T fragment. T r ~ p o n i n ~ ~ - ~ ~ ~ was then made by reconstitution with TnI, TnC, and this COOH-terminal portion of T n T (Fig. 4). If the deleted peptide promoted tropomyosin-tropomyosin interactions, then the cooperativity parameter, y, would be decreased for thin filaments containing troponin70-2Sg instead of whole troponin. Instead (Fig. 5 and Table I), the opposite was found y was slightly greater when T n T residues 1-69 were absent ( y = 82-102) than when the entire TnT molecule was present

TnT 1-259 - TnT 70-259 - Tnl -

TnC - TnT 159-259 -

1 2 3 FIG. 4. Isolation of troponin complexes containing COOH-

terminal fragments of TnT. The identity of troponin70_2s9 and troponin159-?59 was confirmed by SDS-PAGE. Lune I, trop0nin~s9.259. I,ane 2, whole troponin. Lune 3 (from a different gel), troponin?”-259.

h

c - I / - 0.20 - IC T: c T I d ii T: 0.00

I / I

Tn(SO-259)-Tropomyosin, U M 0.00 0 50 1.00 150 2.00 2.50 3.00

FIG. 5. Binding of troponin70-2as-tropomyosin to actin at 300 mM KCl. Binding of troponin,o_259-tropomyosin to actin was assessed under the same conditions (300 mM KC1) as Fig. 3. The nbscissa indicates the concentration of free troponin70-2~g-tropomyosin. Free and bound concentrations were measured as in Fig. 3. The best fit to Equation 1 suggests KO = 26 & 12 X 10:’ M-’ and y = 128 & 62.

( y = 37 for troponin isolated as a native complex, or y = 52- 66 for troponin reconstituted from subunits). Therefore, the value of y for tropomyosin-troponin70-2s9 was intermediate between the values for tropomyosin alone and the values for tropomyosin-whole troponin. In addition, deleting TnT 1-69 caused a modest decrease (about 50%) in the affinity of the troponin-tropomyosin complex for an isolated actin site (KJ . The effect of Ca2+ on t r o p ~ n i n ~ ~ ~ ~ ~ ~ - t r o p o m y o s i n binding to actin was similar to the effect of Ca2+ on troponin-tropomyosin binding to actin; its presence decreased KO by half.

In contrast to the small but detectable effects on y and KO, described above, the overall binding constant yKo was com- pletely unaffected by either reconstitution from troponin subunits or by substitution of T n T 70-259. All of these data suggest that TnT residues 1-69 have only a small effect on thin filament assembly and on interactions between neighboring, regulatory units along the thin filament. However, these experiments do not exclude larger effects on cooperativity under other conditions (see “Discussion”).

Effect of Deletion of TnT Residues 1-158 on Thin Filament Assembly-To further assess the role of different T n T do- mains on interactions among thin filament proteins, troponin lacking TnT residues 1-158 was prepared by chymotryptic digestion of whole troponin (see “Materials and Methods”). This molecule lacks the long NH2-terminal tail of TnT and has weakened affinity for tropomyosin (Morris and Lehrer, 1984). Fig. 6 shows that promoted tropomyosin- actin binding weakly, i.e. only when present in stoichiometric excess and in the absence of Ca2+. Under otherwise identical conditions, troponinlsg-2sg had no effect on tropomyosin-actin binding in the presence of M Ca2+. These results are in qualitative agreement with previous studies showing that binding of the “head” region of troponin to actin and to tropomyosin is inhibited by Ca2+ (Pearlstone and Smillie, 1983). Similarly, our new data suggest that in the absence of T n T residues 1-158, troponin is unable to promote thin filament assembly when Ca2+ is present. The small effect of deletion of TnT residues 1-69 appears greatly magnified by further removal of what could be termed a Ca2+- insensitive anchor, residues 70-158.

Fig. 7 shows representative data permitting quantitative

a 1 1

a l

9 9 0 5 I O ! 5 ? O 2 5 3.0

< 152-?5q) T n , L I ~ !

FIG. 6. Effect of troponinlgs-2gs on tropomyosin-actin binding. Binding of tropomyosin to actin was assessed by Airfuge centrifugation of samples containing the indicated concentration of total trop0nin~sg-2~~ and under conditions (60 mM KCI) identical to Fig. 1, panel A, except 100 p~ CaC12 was present in some samples (circles) and 500 pM dibromo BAPTA was present in others (squares). Samples contained 7 pM actin and 0.3 pM tropomyosin. Little tropomyosin bound to actin a t these concentrations unless troponin159.259 was added. In the presence of CaCl,, however, t r 0 p o n i n ~ ~ ~ . ~ ~ 9 had no effect.


z 0.80 , - E

- i

t 2

c a

h roq s? f -, 4 ..t / - 1 i L 1 u? N i ,/

1 0 4 0 - T $ 1 - C

-T. I i v - + 0.20 - - ,- /- -

” - -/

g 0.00 I/ , 000 020 0 4 G 0 60 0.80 1 0 0 1.20

Tn( 159-?5c?bTropomyosin , uM FIG. 7. Binding of troponin169-26e-tropomyosin to actin.

Binding of troponinla9_259-tropomyosin to actin was assessed by Air- fuge centrifugation of samples under the same conditions as Fig. 1, panel A , except lo-’ M Ca2’ was present. Troponin1s9-259 was always present at 1.5 +M additive excess above the total tropomyosin concentration. Best fit parameter estimation implied KO = 75 & 7.1 x lo3 M-’ and y = 35 k 3.5. Free and actin-bound concentrations of tropomyosin-troponin15g-2s9 were measured as in Fig. 3.

analysis of the assembly of tropomyosin-troponin15g-z59 onto the actin filament in the absence of Ca2+. Because this binding is relatively weak, it was investigated at low ionic strength (60 mM KCl), so the results must be compared to tropomyosin-actin assembly under the same ionic conditions (see Table I). Deletion of TnT residues 1-158 had a much larger effect on KO than did deletion of residues 1-70. increased tropomyosin binding to an isolated site only &fold, compared to the 80-130-fold increase caused by whole troponin and the 25-60-fold increase caused by t r o p ~ n i n ~ ~ _ ~ ~ ~ . Interestingly, the value of y for t r ~ p ~ m y ~ ~ i n - t r ~ p ~ n i n ~ ~ ~ ~ ~ ~ ~ ( y = 32 and y = 35 on two different determinations) was less than the value for tropomyosin alone ( y = 90). This suggests that the tail region of TnT is not required for troponin’s modulatory effect on this cooperativity, which can be attributed primarily to the head region of troponin.

DISCUSSION

This study presents a quantitative analysis of the effect of troponin on tropomyosin binding to an isolated site on F- actin, the effect of troponin on interactions between actin- bound tropomyosin molecules, and the effect of the NH2- terminal region of TnT on these processes. The most surprising result is that troponin’s well-established promotion of tropomyosin-actin assembly is entirely due to the tighter binding of the troponin-tropomyosin complex to an isolated site. Troponin strongly increases tropomyosin viscosity in the absence of actin both for skeletal muscle proteins (Ebashi and Kodama, 1965) and cardiac muscle proteins (Tobacman, 1988). Although this effect is generally attributed to enhanced tropomyosin polymerization, we now report that skeletal muscle troponin modestly decreased the strength of the interactions between cardiac tropomyosin molecules that were bound to the thin filament. We have obtained similar results using a homologous combination of bovine cardiac troponin, tropomyosin, and actin (data not shown).

Because interactions between neighboring tropomyosins promote thin filament assembly, troponin slightly weakens assembly by diminishing the strength of these interactions ( y ) . This weakening is counteracted by the 80-130-fold strengthening of KO (isolated site binding affinity) and, thus,

is not readily apparent in the overall binding expressed by the product yK,. Nevertheless, the decrease in y is significant. Whereas both y and KO concern the process of assembly, y more directly concerns the properties of the assembled filament. Myosin binding (Williams et al., 1988), Ca2+ binding (Tobacman and Sawyer, 1990), and the regulation of tension development (Moss et al., 1985, 1986; Brandt et al., 1987) all appear to be influenced by interactions between regulatory units along the thin filament. The ability of troponin to modulate these interactions may be an important aspect of regulation (Williams et al., 1988; Ishii and Lehrer, 1987; Tobacman and Sawyer, 1990).

Another unexpected result is the small effect of TnT trun- cation on the cooperativity parameter, y. The NHz-terminal region of TnT comprises the long tail region of troponin (Flicker et al., 1982), stretching along the COOH-terminal third of tropomyosin, and reaching beyond the NHz terminus of a neighboring tropomyosin molecule. This has been most clearly demonstrated by x-ray crystallography of troponin and troponin T fragments diffused into tropomyosin crystals. White et al. (1987) showed that the chymotryptic TnT fragment CB3 (residues 1-70) spans the overlapping ends of adjacent tropomyosin molecules. Therefore, this peptide is ideally positioned to alter y. Our studies comparing whole troponin with troponin70-z59 suggest that any such effect is small, but that deleting this peptide may strengthen the interactions between tropomyosins slightly. This approximately 2-fold effect on y must be interpreted with caution: 1) t r o p ~ n i n ~ ~ - ~ ~ ~ contains rat instead of rabbit TnT, and there are five rabbit to rat substitutions within TnT residues 70- 259 (V911, E140D, E195D, N231T, T248A); 2) tropomyosin- t r o p ~ n i n , ~ ~ - ~ ~ ~ yielded a lower value for y than did tropomyosin alone, implying that the tail region of TnT is not required for troponin’s effect on y. Still, the qualitative conclusion from the data is that CB3, the TnT peptide spanning the tropomyosin-tropomyosin overlap joint, does not promote interactions between actin-bound tropomyosins, and may slightly weaken them. This observation reinforces the conclusion (above) that whole troponin modestly decreases y , rather than increases it.

It is mechanistically significant that the effect of troponin on y remains even after a longer section of TnT has been removed (residues 1-158), leaving only the portions of troponin (TnI, TnC, and TnT 159-259) located about 15 nm from the nearest end of the tropomyosin molecule (White et al., 1987). The effect of trop0nin~~9-~59 on y suggests that the COOH-terminal head region of troponin can influence neighboring troponin-tropomyosin complexes, contacted indirectly through actin and/or tropomyosin.

It is unclear how these data can be reconciled with troponin’s ability to promote tropomyosin polymerization. One possibility is that troponin promotes end-to-end contacts between tropomyosins both on and off the thin filament, but only under the lower ionic strength conditions used for viscosity experiments. According to this possibility, the results presented in this paper could be explained as an anomalous effect occurring only at high ionic strength. However, this would not explain other puzzling results. For example, high ionic strength is known to lessen viscosity, yet tropomyosin- tropomyosin interactions in the presence of actin as measured by y increased or remained the same from 60 to 300 mM KC1 (Table I, Fig. 1). Other incongruous results have been reported in the literature. Phosphorylation of tropomyosin, for example, increases its viscosity, but has only a negligible effect on the cooperative tropomyosin-actin binding (Heeley et al., 1989). Carboxypeptidase-treated tropomyosin and NH2-ter-


minal unacetylated tropomyosin do not polymerize but will bind to actin cooperatively in the presence of troponin (Hee- ley, et al., 1987, Hitchcock-DeGregori and Heald, 1987, Heald and Hitchcock-DeGregori, 1988). Also, the chymotryptic fragment troponin T (1-159) is reported to increase tropomyosin viscosity (Jackson et al., 1975), but will not induce tropomyosin actin binding (Heeley et al., 1987). Nor would discounting our results explain the problematic pattern of the troponin- induced increase in tropomyosin viscosity: the viscosity does not saturate, but instead continues to rise as superstoichiom- etric concentrations of troponin are added (Tobacman, 1988). The simplest explanation for these apparent discrepancies is that viscosity measurements performed in the absence of actin do not reflect the strength of interactions between neighboring, actin-bound tropomyosins (or troponin-tropomyosins). This may be because these interactions are very different when the complexes are bound to actin, or it could merely reflect the qualitative nature of the viscosity measurements. In either case, viscosity appears to measure a different prop- erty than the cooperativity parameter that promotes tropomyosin-actin binding.

In contrast to the unexpected results concerning cooperativity, our data on how KO is influenced by troponin, Ca2+, and various regions of TnT are easy to reconcile with prior models for thin filament assembly. Troponin increases KO for tropomyosin-actin binding 80-130-fold. Adding Ca2+ weakens KO, the binding of troponin-tropomyosin to actin, although only by half. Removal of T n T residues 1-69 slightly diminished KO so that it is only 60-fold greater than for tropomyosin-actin binding, and it also did not eliminate the effect of Ca’+. When TnT was further truncated, however, the remain- ing tropomyosin-troponinl~~~25~ complex bound much more weakly to actin; in the absence of T n T residues 1-158, troponin enhanced tropomyosin-actin binding only 6-fold. Fur- thermore, T n T residues 70-158 provide an essential Ca’+- insensitive anchor as described by Pearlstone and Smillie (1983), since t r o p ~ n i n , ~ ~ - ~ ~ ~ promoted tropomyosin-actin binding only in the absence of Ca2+. In the presence of Ca2+ we detected no effect of on tropomyosin-actin binding either because it failed to bind to tropomyosin, or because tropomyosin and tropomyosin-troponin15g-2sg bind in- distinguishably to actin. It should be mentioned that Heeley et al. (1987) used unreconstituted subunits and did detect minimal binding between troponin T fragment (159-259), tropomyosin, and actin in the presence of Ca+*. All of these results are consistent with a two-site model, similar to that previously proposed (Heeley et al., 1987): 1) Ca2+-regulated (i.e. inhibited) binding of TnI to actin and the COOH-terminal region of TnT to tropomyosin, and 2) Ca’+-insensitive promotion of troponin-tropomyosin binding to actin by T n T

An important assumption in our analysis is that the tropomyosin or troponin-tropomyosin is monomeric when not bound to actin. Sedimentation equilibrium data support this assumption for tropomyosin alone (Wegner, 1979), but we have no such proof for the troponin-tropomyosin complex. For several reasons, however, polymerization of troponin- tropomyosin was unlikely to have falsified the results: 1) troponin-tropomyosin saturated the thin filament at low protein concentrations (less than 1 p ~ ) which would inhibit polymerization, especially a t high ionic strength; 2) qualitatively similar values for y were obtained under a variety of conditions and with a variety of troponin complexes (Table I). Furthermore, replacing the tropomyosin with unacetylated tropomyosin, which, according to viscosity assays, does not polymerize in the presence of troponin (Hitchcock et al., 1987)

(70-158).

also has little effect on y.’ Nevertheless, we cannot prove that all of the troponin-tropomyosin was monomeric, and there remains a possibility that measurements of KO and y were affected.

I t is pertinent to compare our data to previous reports of thin filament assembly. The same linear lattice model was used in one earlier study of tropomyosin-actin binding, with results implying much higher levels of cooperativity (Wegner, 1979). Values of y obtained in the absence of troponin were as high as 1000, much greater than values found under any conditions in the present study. In a subsequent study by the same investigator under different experimental conditions (Wegner and Walsh, 1981), the value of y for the troponin- tropomyosin complex was determined to be about 200, lower than the above-mentioned value for tropomyosin alone, but higher than now reported for troponin-tropomyosin. It should be emphasized that while these earlier values are greater than those in Table I, Wegner’s data were consistent with the trend now noted in this report, the presence of troponin decreased Y.

A more recent publication (Heeley et al., 1989) employing conditions very similar to those now described (60 mM KCl, 2.5 mM MgC12) supports the accuracy of our measurements of y. Applying Equation 1 to data taken from Fig. 2C in this earlier report, we calculate y to equal 68 f 11 in the absence of troponin (data not shown), in good agreement with the present study. This previously published experiment also implied that yKo equaled 4 x lo6 M-’, according to our calcu- lations, more than 2-fold tighter than we now report. There- fore, we cannot exclude the possibility that the carboxymethylated tropomyosin used in our investigation has somewhat altered actin affinity. However, experiments mixing labeled and unlabeled tropomyosin (see “Materials and Methods”) detected no such effect.

Our results are in general agreement with a recent publication by Pan et al. (1991) concerning another T n T fragment, TnT 46-259. Deletion of 45 NH2-terminal residues of TnT slightly strengthened, rather than weakened, the affinity of T n T for immobilized tropomyosin. Troponin complexes containing T n T 46-259 bound to actin-tropomyosin and con- ferred Ca2+-sensitive regulation on the MgATPase rate of myosin S-1. Pan et al.’s data, our previous studies with cardiac T n T (Tobacman, 1987), Ohtsuki’s experiments with TnT 46- 259 (1984), and the current results all indicate that the NHz terminus of T n T is not required for many aspects of T n T function. However, all of these studies found subtle effects attributable to this region of TnT, implying that it has a modulatory role, and perhaps explaining its complex regulation by alternative RNA processing (Breitbart et al., 1985). Furthermore, none of these reports exclude an effect of the TnT NHz-terminal region on cooperative phenomena that depend on actin-myosin binding. It is hoped that future research into the structure and assembly of the thin filament will allow further elucidation of these processes.

Acknowledgments-We express our gratitude to Drs. Nadal-Ginard and Breitbart for providing us with the TnT cDNA and to Dr. Daniel Weeks for helping us with the initial stages of the recombinant DNA experiments.

REFERENCES

Bradford M. M. (1976) Anal. Eiochem. 72,248-254 Brandt. b. W.. Diamond. M. S.. Rutchik. J. S.. and Schachat. F. H. (lQ*71 -1 Ma. bid. 195,885-896

and Nadal-Ginard, B. (1985) Cell 41.67-82

ll., I.

Breitbart, R. E., Nguyen, H. T., Medford, R. M., Destree, A. T., Mahdavi, V.,

L. S. Tobacman, K. Willadsen, L. E. Hill, J. P. Mehegan, and C. A. Butters, manuscript in preparation.


Cho, Y . J., Liu, J., and Hitchcock-DeGregori. S. E. (1990) J. Biol. Chem. 2 6 5 ,

Davis, L. G., Dibner. M. B.. and Battey, J. F. (1986) Basic Methods in Molecular 538-545

Ebashi. S.. and Kodama. A. (1965) Et Biology, Elsevier, New York

Hie?&, D. H., Golosinska, K., and Smillie, L. B. (1987) J. Biol. Chem. 2 6 2 ,

Heeley, D. H., Watson, M. H., Mak, A. S., Dubord, P., and Smillie, L. B. (1989)

Herzberg, O., and James, M. N. (1988) J. Mol. Biol. 203,761-779 Hill, T. L. (1983) Biophys. J. 4 4 , 383-396 Hill, T. L. (1985) Cooperatiuity Theory in Biochemistry, Springer-Verlag, New

Hill, T. L., Eisenberg, E., and Greene, L. E. (1980) Proc. Natl. Acad. Sci. U. S. A .

Hill, T. L., Eisenberg, E., and Greene, L. E. (1983) Proc. Natl. Acad. Sci. U. S. A.

Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J . Biol. Chem. 262,9730-

Hitchcock-DeGregori, S. E., and Varnell, T. A. (1990) J. Mol. Bid. 2 1 4 , 885-

Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 3 4 7 ,

Huxley, H. E. (1972) Cold Spring Harbor Symp. Quant. Bid. 37,341-352 Ingraham, R. H., and Swenson, C. A. (1984) J. Bid. Chem. 259,9544-9548 Ingraham, R. H., and Swenson, C. A. (1985) Biochemistry 24,5221-5225

Ishii, Y., and Lehrer, S. S. (1991) J. Biol. Chem. 2 6 6 , 6894-6903 Ishii, Y., and Lehrer, S. S. (1987) Biochemistry 26,4922-4925

Jackson, P., Amphlett, G. W., and Perry, S. V. (1975) Biochem. J. 161,85-97 Mak, A. S., Golosinska, K., and Smillie, L. B. (1983) J. Biol. Chem. 258 ,

Maniatis, T., Fritscb, E. F., and Sambrook, J. (1982) Molecular Cloning: A

9971-9978

J. Bid. Chem. 264,2424-2430

York

77,3186-3190

8 0 , 60-64

9735

896

44-49

14330-14334

Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

McGhee, J. D., and von Hippel, P. H. (1974) J. Mol. Biol. 86,469-489

Milligan, R. A,, Whittaker, M., and Safer, D. (1990) Nature 348,217-221 Mehegan, J. P., and Tobacman, L. S. (1991) J . Biol. Chem. 266,966-972

Moss, R. L., Giulian, G. G., and Greaser, M. L. (1985) J. Gen. Physlol. 86,585- Morris, E. P., and Lehrer, S. S. (1984) Biochemistry 2 3 , 2214-2220

Moss, R. L., Allen, J. D., and Greaser, M. L. (1986) J. Gen. Physiol. 8 7 , 761- 600

774

Ohtsuki, I., Shiraishi, F., Suenaga, N., Miyata, T., and Tanokura, M. (1984) J .

Pan, B . 3 . Gordon, A. M., and Potter, J. D. (1991) J. Biol. Chem. 266,12432- Biochem. (Tokyo) 9 5 , 1337-1342

12438 Pearlstone, J. R., and Smillie, L. B. (1983) J. Biol. Chem. 258,2534-2542 Phillips, G. N, Fillers, J. P., and Cohen, C. (1986) J. Mol. Bid. 192 , 111-131 Potter, J. D. (1982) Methods Enzymol. 85,241-263 Potter, J. D., and Gergely, J. (1974) Biochemistrv 13 , 2697-2703 Sanger, F., Nicklen, S., and Coulson, A. S. (1977") Proc Natl. Acad. Sci. U. S. A.

Spudich, J. A,, and Watt, S. (1971) J. Bid. Chem. 2 4 6 , 4866-4871 Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990)

Tobacman, L. (1987) Biochemistry 26,492-497 Tobacman, L. S. (1988) J. Biol. Chem. 263,2668-2672 Tobacman, L. S., and Adelstein, R. S. (1986) Biochemistry 25 , 798-802

Tobacman, L. S., and Sawyer, D. (1990) J. Bid. Chem. 265,931-939 Tobacman, L. S., and Lee, R. (1987) J . Biol. Chem. 262,4059-4064

Wegner, A. (1979) J. Mol. Bid. 131,839-853 Wegner, A,, and Walsh, T. P. (1981) Biochemistry 20 , 5633-5642 White, S. P., Cohen, C., and Phillips, G. N. (1987) Nature 325,826-828 Williams, D. L., Greene, L. E., and Eisenberg, E., (1988) Biochemistry 27,

74,5463-5467

Methods Enz mol 185,60-89

Zasedatelev. A. S., Gurskii, G. V., and Vol'kenshtein. M. V. (1971) Mol. Biol. 6987-6993

5 , 245-251

Analysis of Troponin-Tropomyosin Binding to Actin

Documents

Transcript of Analysis of Troponin-Tropomyosin Binding to Actin