©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping the Functional Domains within the Carboxyl Terminus of -Tropomyosin Encoded by the Alternatively Spliced Ninth Exon (*)

(Received for publication, August 25, 1995; and in revised form, December 16, 1995)

Robin L. Hammell (§) Sarah E. Hitchcock-DeGregori

From the Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tropomyosins are highly conserved, coiled-coil actin binding proteins found in most eukaryotic cells. Striated and smooth muscle alpha-tropomyosins differ by the regions encoded by exons 2 and 9. Unacetylated smooth tropomyosin expressed in Escherichia coli binds actin with high affinity, whereas unacetylated striated tropomyosin requires troponin, found only in striated muscle, for strong actin binding. The residues encoded by exon 9 cause these differences (Cho, Y.-J., and Hitchcock-DeGregori, S. E.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10153-10157). We mapped the functional domains encoded by the alpha-tropomyosin exon 9a (striated muscle-specific) and 9d (constitutively expressed), by measuring actin binding and regulation of the actomyosin MgATPase by tropomyosin exon 9 chimeras and truncation mutants expressed in E. coli. We have shown that: 1) the carboxyl-terminal nine residues define the actin affinity of unacetylated tropomyosin; 2) in the presence of Ca, the entire exon 9a is required for troponin to promote fully high affinity actin binding; 3) the first 18 residues encoded by exon 9a are critical for the interaction of troponin with tropomyosin on the thin filament, even in the absence of Ca. The results give new insight into the structural requirements of tropomyosin for thin filament assembly and regulatory function.


INTRODUCTION

Tropomyosin (TM) (^1)is an actin binding protein, first discovered in skeletal muscle(1) , and now known to be present in virtually all eukaryotic cells. Tropomyosins are a family of highly conserved, coiled-coil proteins in which diversity is primarily accomplished through the use of alternative promoters and alternative RNA splicing of the transcripts of a small number of genes (three to four in vertebrates; reviewed in (2) ).

Tropomyosins are expressed in developmental and tissue-specific patterns. Certain isoforms are found in more than one cell type and tissue, although they may have different localizations within cells (e.g.(3) ). Other TM isoforms are specific, notably those expressed in striated and smooth muscles (striated and beta, smooth) and the specific brain alpha-TM isoforms ( (4) and (5) ; reviewed in (2) ). These isoforms contain uniquely-expressed exons that presumably encode domains of the protein involved in isoform-specific function.

The functions of TM are best understood from the extensive in vitro biochemical and biophysical analysis since the protein's discovery. Two fundamental functions that define TMs are cooperative binding to F-actin (6) and cooperative inhibition or activation of the actomyosin MgATPase(7) . Tropomyosin isoforms differ in these assays (reviewed in (8) ), and their activities are influenced by other cytoskeletal proteins that interact directly or indirectly with TM (caldesmon, troponin (Tn), and myosin).

The cellular function of TM is best understood in striated muscles where, through interaction with Tn, it regulates contraction in a Ca-dependent manner (reviewed in (9, 10, 11) ). Biochemical and structural studies by numerous investigators have led to the following model of the thin filament. Tropomyosin molecules are aligned end-to-end (NH(2) terminus to COOH terminus) in the grooves of the helical actin filament(12, 13) . Troponin is bound to TM along the COOH-terminal third of the molecule (14, 15) . Troponin I, TnC, and the COOH terminus of TnT are positioned near Cys-190 of TM. The elongated NH(2) terminus of TnT extends toward the COOH terminus of TM to the beginning of the next TM along the actin filament. Troponin greatly increases the affinity of TM for actin in the presence of Ca, with a further increase upon removal of Ca(16) . Based on binding studies with TM and Tn peptides, Mak and Smillie (17) proposed that the Tn-TM interaction in the region of Cys-190 is Ca-sensitive (stronger in the absence of Ca), whereas that at the COOH terminus and the overlap region is Ca-independent (strong both in the presence and absence of Ca). Substantial evidence has since supported this model.

The Ca-independent TnT binding site on TM includes the COOH-terminal 27 amino acids encoded by the striated muscle-specific exon 9a, implying a specific role in Tn-dependent regulatory function. Exon 9a is uniquely expressed in the striated isoform whereas the COOH-terminal coding exon of most other alpha-TM isoforms, is exon 9d (18; reviewed in (2) ). The functional differences between these 284 residue alpha-TMs are primarily attributable to the exon 9-encoded COOH terminus(19) . Unacetylated striated alpha-TM expressed in Escherichia coli binds poorly to F-actin because the NH(2)-terminal Met is unacetylated (20, 21, 22) whereas TMs encoded by cDNAs containing exon 9d (smooth and TM2, a non-muscle isoform) bind well(19) . In the presence of Ca, Tn greatly increases the affinity of the unacetylated striated TM for actin but has little effect on unacetylated smooth TM or TM2(19) . Similar differences have been reported between unacetylated smooth and striated beta-TMs(23, 24) .

In the present work we have defined the functional domains encoded by alpha-TM exons 9a and 9d (two of the four COOH-terminal alpha-TM exons) by making a series of TM chimeras and truncation mutants. Unacetylated TMs expressed in E. coli offer a powerful system for looking at gain or loss of TM functions: actin affinity and Ca-independent interaction with Tn on the actin filament. We have shown that: 1) the actin affinity of unacetylated TM is defined by the COOH-terminal nine amino acids; 2) the entire striated-specific exon 9a is required for Tn to promote fully high affinity binding of TM to actin in the presence of Ca; and 3) the first 18 residues encoded by exon 9a are critical for the interaction of Tn with TM on the thin filament, even in the absence of Ca. The results, together with previous work from our laboratory and others, indicate that the NH(2)- and COOH-terminal ends of TM are primary functional determinants. The results are discussed in terms of the structural requirements of TM for regulatory function with Tn. Portions of this work have been reported in a preliminary form (25) .


MATERIALS AND METHODS

DNA Constructions and Purification of Proteins

General recombinant DNA techniques were performed as described by Sambrook et al.(26) or as recommended by the supplier.

Rat striated (pUC18/TM9a) and rat smooth (pBR322/SMTM) muscle alpha-TM cDNA clones were the gift of B. Nadal-Ginard(4) . The coding regions differ only in exons 2 and 9 corresponding to codons 39-80 and codons 258-284, respectively, with codon number 1 defined as the ATG immediately following the NcoI cleavage site.

To facilitate a subsequent cloning step, both striated and smooth cDNAs were subcloned into pUC119, destroying the vector's SmaI site (Fig. 1A). In order to accomplish this, TM9a cDNA was digested with EcoRI. The 1212-base pair fragment was isolated from an agarose gel, after which the 5` overhang was filled in using Klenow. This blunt fragment was ligated to the SmaI site of pUC119 resulting in the vector, pUC119/TM9a. The smooth tropomyosin cDNA was digested with PstI. The 1078-base pair fragment was isolated from an agarose gel, after which the 3` overhangs were filled in using T4 DNA polymerase. This blunt fragment was ligated to the SmaI site of pUC119 resulting in the vector, pUC119/SMTM. The NcoI-SmaI fragment from pUC119/TM9a containing TM codons 1-237 was ligated to the NcoI-SmaI fragment from pUC119/SMTM containing TM codons 238-284. The resulting chimera is pUC119/TM9d.


Figure 1: Construction of tropomyosin exon 9 variant plasmids (see ``Materials and Methods'' for details). A, striated (TM9a) and smooth (SMTM) cDNAs were subcloned into pUC119 in order facilitate future cloning steps. The NcoI-SmaI fragment encoding TM9a codons 1-237 was ligated to the NcoI-SmaI fragment encoding SMTM codons 238-284 resulting in TM9d. B, to generate TMs with chimeric ninth exons (TM9a/9d and TM9d/9a), the EcoRI-AvaII fragments of TM9a and TM9d containing TM codons 1-275 and the AvaII-BamHI fragments of TM9a and TM9d containing codons 276-284 were exchanged then ligated with pUC119. C, tropomyosin truncation mutants with the 3` nine codons deleted were constructed by filling in the 5` overhangs of the EcoRI-AvaII fragment (B) with Klenow then ligating to a BamHI linker. After digesting with NcoI and BamHI, this fragment was ligated to the expression vector, pET11d(35) .



To construct TMs which contain chimeric ninth exons (TM9a/9d, TM9d/9a), pUC119/TM9a and pUC119/TM9d were digested with AvaII (Fig. 1B). The fragments containing TM codons 1-275 were further digested with EcoRI, and the fragments containing TM codons 276-284 were further digested with BamHI. The fragments containing TM codons 1-275 and TM codons 276-284 of pUC119/TM9a and pUC119/TM9d were exchanged and then ligated with pUC119 which had been linearized with EcoRI and BamHI. The result is pUC119/TM9a/9d and pUC119/TM9d/9a.

Truncation mutants were constructed by deleting the cDNA coding for the carboxyl-terminal 9 residues of TM (Fig. 1C). This was accomplished by filling in the 5` overhang of the AvaII fragment isolated from pUC119/TM9a using Klenow and ligating it to a linker (TGATAAGGATCCTTATCA) containing termination codons (underlined) followed by a BamHI site (in italics). After digestion with NcoI and BamHI, the fragment was ligated to the NcoI and BamHI sites of the pET11d expression vector (27) resulting in pET11d/TM9a/. pUC119/TM9d was treated in the same manner resulting in pET11d/TM9d/.

The NcoI-BamHI fragments of pUC119/TM9a, pUC119/TM9d, pUC119/TM9d/9a, and pUC119/TM9a/9d were ligated to the NcoI and BamHI sites of the expression vector, pET11d. The cDNAs for all six TM variants in pET11d were expressed in E. coli (BL21{DE3}pLysS for TM9a or BL21{DE3} for the other TM variants)(27) . The TMs were purified from 2 liters of cells (yield = 20-30 mg/liter) as described previously (20) except the (NH(4))(2)SO(4) fractionation was 35-70% saturation instead of 35-60%.

Actin was purified from acetone powder of pectoral muscle of White Leghorn chickens according to (28) , except actin was polymerized by addition of KCl and MgCl(2) to a final concentration of 20 and 0.7 mM, respectively, and incubated at 37 °C for 10 min before cooling slowly to room temperature.

Troponin was purified from pectoral muscle pulp of White Leghorn chickens obtained from Dr. J. Fagan (Rutgers University, NJ) using the procedure of Potter (29) except, alternate Triton X-100 extractions included the following protease inhibitors: 2 mM E64, 1 mg/ml leupeptin; 2 mM 4-amidinophenylmethane-sulfonyl fluoride, 0.5 M benzamidine. Also, the (NH(4))(2)SO(4) crude Tn pellet was dialyzed against 20 mM Tris, pH 7.5, 0.1 mM CaCl(2), and 0.5 mM dithiothreitol (DTT) then purified using ion exchange (DE52) column chromatography with a 0-0.6 M NaCl gradient. Chicken pectoral muscle myosin was isolated using the method of Margossian and Lowey(30) .

Modified Recombinant Tropomyosin Purification Method (31

Recombinant striated alpha-TM (TM9a) was purified to the point of heat denaturation as described by Fanning et al.(31) . This method is based on the procedure of Hitchcock-DeGregori and Heald (20) with two major modifications: 1) the clarified cell extract was subjected to three isoelectric precipitations prior to (NH(4))(2)SO(4) fractionation (45-65% saturation instead of 35-70%) and 2) the boiling step was performed in low ionic strength buffer (50 mM instead of 1 M). A sample of the heat stable supernatant was dialyzed against 10 mM imidazole, pH 7.0, 5 mM MgCl(2), 1 mM DTT, 0.1 mM EGTA containing 100 mM NaCl and assayed for the ability to bind actin in the same buffer.

Conformational Analysis

Circular dichroism spectroscopy was used to measure the folding of TM as a function of temperature in 500 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, and 1 mM DTT(32) . The TM exon 9 variants showed multiple transitions with virtually the same degree of folding at 20 °C(33) . The sequence of the COOH-terminal 27 residues has a small effect on the overall thermal stability of TM: the first 18 residues of exon 9a are associated with a slight increase in stability (2 °C) whereas the last nine result in a slight decrease (Table 1). These small differences can be understood in terms of changes in the hydrophobicity of the a and d residues of the heptad repeat that forms the interface between the two alpha-helices of the coiled coil. It has not been possible to relate actin affinity to the thermal stability of these or other recombinant TMs.



Determination of Protein Concentration

Protein concentration was determined by measuring the difference absorption spectrum at 295 nm in 6 M guanidine HCl, 50 mM phosphate buffer, pH 12.5 (where all the tyrosines are ionized), versus 6 M guanidine HCl, 50 mM phosphate buffer, pH 6, in a reference beam spectrophotometer(34) . The concentration was calculated assuming a value 295(Tyr) = 2480 M cm(35) and 8 Tyr/mol (TM9d, TM9d/, TM9d/9a) or 12 Tyr/mol (TM9a, TM9a/, TM9a/9d). The concentration of the TM used for the standard curve for densitometry was determined using a micro-biuret assay using bovine serum albumin as a standard (36) and confirmed using the above spectrophotometric assay. The concentration of actin, myosin, and Tn was determined spectrophotometrically using the extinction coefficients (1% at 280 nm) 11.0, 5.3, and 4.5, respectively.

Actin Binding Assays

Binding of TM to actin was measured by cosedimentation as described(37) , except that the centrifugation was for 25 min at 60,000 rpm in a Beckman model TL-100 ultracentrifuge in a TLA-100 rotor. Tropomyosin and Tn (2 µM) were cosedimented with actin (5 µM) at 25 °C in 150 mM NaCl, 10 mM Tris, pH 7.5, 2 mM MgCl(2), 0.5 mM DTT, 0.2 mM CaCl(2) or 0.2 mM EGTA. The pellets and supernatants were analyzed electrophoretically on 10% SDS-polyacrylamide gels(38) . Protein was visualized using Coomassie Blue and quantitated using a Molecular Dynamics model 300A computing densitometer(21) . The concentration of free TM in the supernatant was determined using a standard curve prepared with known amounts of recombinant smooth alpha-TM. Bovine serum albumin was included in the sample buffer as an internal loading standard to correct for variations in sample loading. Apparent binding constants (K) and Hill coefficients (alpha(H)) were determined using SigmaPlot (Jandel Scientific) to fit the data to the equation:

Actomyosin MgATPase Assay

The actomyosin MgATPase was measured as a function of TM concentration using 2.4 µM actin, 0.6 µM myosin, 0-0.6 µM TM, 1 µM Tn in 40 mM NaCl, 5 mM imidazole, pH 7.0, 0.5 mM MgCl(2), 5 mM MgATP, 1 mM DTT, 0.2 mM EGTA, or 0.2 mM CaCl(2). Assays, with a final volume of 75 µl, were carried out in 96-well microtiter dishes at 28 °C in a thermoequilibrated Molecular Devices ThermoMax microtiter plate reader. The reaction was initiated by the addition of MgATP to a final concentration of 5 mM and terminated by the addition of 25 µl of 13.4% SDS, 0.12 M EDTA after 15 min. The amount of inorganic phosphate released was determined colorimetrically according to White (39) and the plates were read in a Molecular Devices ThermoMax microtiter plate reader with a 650-nm filter. Time courses had been carried out previously to show that phosphate liberation was linear. The assays were single point determinations. Specific activity is expressed as micromole of P(i)/mg of myosin/min.


RESULTS

The isoform-specific differences in actin affinity and interaction with Tn of recombinant, unacetylated 284 residue alpha-TMs can be primarily attributed to differences in residues 258-287, encoded by exon 9(19) . To investigate the regions encoded by exons 9a and 9d (18; reviewed in (2) ) that give rise to the isoform-specific function, we made a series of chimeras and deletion mutants, illustrated in Fig. 2A, in which the last nine residues have been exchanged or deleted. The sequences of the variants are identical except for the residues encoded by the two ninth exons, shown in Fig. 2B. The deletion mutant TM9a/ is similar to carboxypeptidase-digested TM in which the last 11 residues are removed enzymatically. These six variants were expressed in E. coli as unacetylated TMs which offer the opportunity to study gain or loss of TM-specific functions: high affinity actin binding, the ability of Tn to increase the affinity of TM for actin, and Ca regulation of the actomyosin ATPase with Tn.


Figure 2: Tropomyosin exon 9 variants. A, all variants are identical to striated rat alpha-TM except for changes in the ninth exon represented by boxes. Numbers indicate the amino acid position at the boundaries of the changes. B, comparison of rat alpha-TM exon 9a and 9d encoded sequences. Identities are enclosed in boxes; the positions in the heptapeptad repeat are shown in lower case letters.



Actin Binding of Tropomyosin Exon 9 Variants

The actin affinity of the TM exon 9 variants was measured by cosedimentation (Fig. 3, Table 2; 21, 37). TM9a (striated alpha-TM) expressed in E. coli bound poorly to actin, as previously reported, due to the unacetylated NH(2) terminus(19, 20, 21, 22, 37) . In contrast, TM9d (alpha-TM2, a non-muscle isoform) bound well, as do other TMs containing this COOH-terminal sequence(19, 24, 40, 41) . TM9a/9d, a chimera containing the last nine residues encoded by exon 9d, bound to actin with the same affinity as TM9d whereas TM9d/9a, with the last nine residues encoded by exon 9a, bound as weakly as did TM9a. The actin affinity of the TMs lacking the COOH-terminal nine residues (TM9a/ and TM9d/) was too low to measure using this assay. These results clearly show that the last nine residues encoded by exon 9 define the actin affinity of unacetylated recombinant alpha-TM.


Figure 3: Actin binding of tropomyosin exon 9 variants. Conditions: TM was cosedimented with actin (5 µM) at 25 °C in 150 mM NaCl, 10 mM Tris, pH 7.5, 2 mM MgCl(2), 0.5 mM DTT. bullet, TM9a/9d; box, TM9d; circle, TM9a; , TM9d/9a; , TM9d/; Delta, TM9a/. The TM/actin ratio determined using densitometry (arbitrary units) was normalized using the n reported by SigmaPlot using the Hill equation (see ``Materials and Methods''). The data shown are from three experiments for TMs which bind (TM9a/9d, TM9d) and from one to two experiments for TMs which do not bind (TM9a, TM9d/9a, TM9d/, TM9a/).





Fanning et al.(31) reported that unacetylated striated alpha-TM purified using a modified method (see ``Materials and Methods'') binds actin strongly in the presence of 100 mM KCl. However, we found that unacetylated striated alpha-TM purified by the Fanning method (31) bound poorly to actin in 100 mM NaCl (K(a) < 1 times 10^5M) whereas the same protein purified using the Hitchcock-DeGregori method (20) bound with measurable affinity (K(a) 1 times 10^6M), suggesting that the modified purification method adversely affects TM function. Also, acetylated muscle TM repurified using the Hitchcock-DeGregori method (20) bound actin with high affinity, indicating that this method does not alter the TM itself. These results confirm that the weaker actin affinity of striated alpha-TM purified from E. coli is a consequence of the lack of NH(2)-terminal acetylation and not the purification method(20) .

Actin Binding of Tropomyosin Exon 9 Variants in the Presence of Troponin

As with muscle (acetylated) TM, Tn increases the affinity of unacetylated striated alpha-TM (TM9a) for actin whereas it has little effect on the affinity of smooth TM or TM9d for actin(19, 37) . The results in Fig. 4A and Table 2show that the complete exon 9a is required for Tn to result in the maximal increase in actin affinity in the presence of Ca. Troponin increased the affinity of TM9a about 100-fold, whereas the increases were 4-fold for TM9a/9d and about 5-10-fold for TM9d/9a, both greater than TM9d (1.5-fold). In the presence of Tn and Ca the TM variants containing the first 18 residues encoded by exon 9a (TM9a, TM9a/9d) bound to actin with lower cooperativity than TM9d (Fig. 4A, Table 2). We infer that both regions encoded by the striated muscle-specific exon 9a are critical for Tn-TM interaction with the actin filament in the presence of Ca. The results are consistent with the Mak and Smillie model (17) that the COOH terminus is the binding site of Tn on TM in the activated (+Ca) state (the Ca-independent site). Neither truncation mutant, TM9a/ or TM9d/, bound in the conditions of this assay, implying that a full-length TM is required.


Figure 4: Actin binding of tropomyosin exon 9 variants with Tn in the presence (A) and absence (B) of calcium. Conditions: TM and Tn (2 µM) were cosedimented with actin (5 µM) at 25 °C in 150 mM NaCl, 10 mM Tris, pH 7.5, 2 mM MgCl(2), 0.5 mM DTT, 0.2 mM CaCl(2), or 0.2 mM EGTA. bullet, TM9a/9d; box, TM9d; circle, TM9a; , TM9d/9a; , TM9d/; Delta, TM9a/. The TM/actin ratio determined using densitometry (arbitrary units) was normalized using the n reported by SigmaPlot using the Hill equation (see ``Materials and Methods''). The data shown in A are from three experiments for TMs which bind (TM9a/9d, TM9d, TM9a, TM9d/9a) and from one to two experiments for TMs which do not bind (TM9d/, TM9a/). In B, data are from two experiments for TMs which bind (TM9d/9a, TM9a/) and from one experiment for TMs which bind too tightly to measure (TM9a/9d, TM9d, and TM9a) or do not bind (TM9d/).



With Tn in the absence of Ca, the affinity of all TM exon 9 variants was greater, with the exception of TM9d/ (Fig. 4B, Table 2). The increase in affinity was in the order of 10-fold, consistent with our previous measurements(19) , although certain variants bound too tightly to measure accurately in the conditions of these experiments. TM9a/, whose affinity was too weak to measure in the presence Ca, bound well in the absence of Ca, as previously reported for carboxypeptidase-treated unacetylated TMs(42) .

Most interesting, however, is that TM9d/ did not bind to actin, even in the absence of Ca while TM9a/ did. Since the Cys-190 region, which includes the Ca-sensitive binding site of Tn on TM (17) is identical in all exon 9 variants studied, we expected that Tn-TM interaction in the absence of Ca would be the same in TM9a/ and TM9d/. Clearly, the binding of Tn only to the Cys-190 region of TM is insufficient to assemble Tn-TM on the actin filament, even in the absence of Ca. The results suggest that the first 18 residues of exon 9a are important for the increased affinity of Tn-TM for actin in the absence as well as in the presence of Ca.

The binding constants for TM9a and TM9d reported here are higher than those previously published(19) . The present TMs have been expressed using a different expression system with higher yields and better purity. We have experienced similar measurements of increased affinity in other recombinant TMs. (^2)

Calcium Regulation of the Actomyosin MgATPase by Tropomyosin Exon 9 Variants with Troponin

The regulatory function of the TM exon 9 variants was evaluated by measuring their ability to confer calcium-dependent regulation of the actomyosin MgATPase with Tn. All TM variants that bound to actin with Tn inhibited the actomyosin ATPase in a Ca-sensitive manner with similar effectiveness (Fig. 5). In contrast, TM9d/, which did not bind to actin in the conditions of the ATPase assay, did not regulate. In the presence of Ca, the ATPase activity was the same with all variants. For simplicity, only TM9a is shown in Fig. 5. These results are consistent with previous reports from this laboratory that TMs that can bind to actin with Tn are similar in their ability to regulate the actomyosin ATPase(19, 43) .


Figure 5: Effect of tropomyosin exon 9 variants on the actomyosin Mg ATPase in the presence of Tn without Ca. Conditions: 2.4 µM actin, 0.6 µM myosin, 0-0.6 µM TM, 1 µM Tn in 40 mM NaCl, 5 mM imidazole, pH 7.0, 0.5 mM MgCl(2), 5 mM MgATP, 1 mM DTT, 0.2 mM EGTA for 15 min at 28 °C. Specific activity is expressed as micromoles of P(i)/mg of myosin/min. In the absence of TM, the mean specific activity of the actin-Tn-myosin ATPase for all experiments pooled was 0.15 ± 0.01 µmol of P(i)/mg of myosin/min (range = 0.13-0.17 µmol of P(i)/mg of myosin/min; n = 26). Each curve contains normalized data pooled from two to five experiments. The data were normalized to the mean specific activity of the actin-Tn-myosin ATPase in the absence of TM for each experiment. Only TM9a is shown with Tn and 0.2 mM Ca, since it is representative of all the TM exon 9 variants.




DISCUSSION

In the present work we have defined the functional domains encoded by exon 9a, a striated muscle-specific exon, and exon 9d, the constitutively-expressed exon found in smooth and most non-muscle alpha-TMs. The COOH-terminal nine amino acids of TM9d allow unacetylated TM to bind to actin with high affinity. The exon 9a-encoded COOH terminus, especially the first 18 residues, confers an isoform-specific binding site for Tn which is essential for Tn-dependent thin filament assembly of unacetylated TM. The COOH-terminal domain is the most variable among TM isoforms; it is encoded by alternative exons (four in alpha-TMs) and it is more variable between genes and species than much of the rest of TM(2, 44) .

We have shown that the last nine amino acids are fully responsible for the differences in actin affinity (without Tn) between unacetylated TM9a (striated TM; low affinity for actin) and TM9d (TM2, a non-muscle isoform; high affinity for actin). Comparison of the amino acid sequences of the last nine residues encoded by exons 9a and 9d (Fig. 2B) shows that there is only one identity (Leu-278) and three conservative changes. At this point it is difficult to predict the involvement of specific residues in the observed changes in actin affinity. Interestingly, Pittenger et al.(45) reported that striated beta-TM (exon 9a) binds to actin with higher affinity than smooth beta-TM (exon 9d) although the amino acid sequences of the last nine residues encoded by beta-TM exons 9a and 9d differ from the corresponding alpha-TM ninth exons by only one to two conservative changes.

Our second major conclusion is that the entire striated-specific exon 9a is required for Tn to promote fully high affinity binding of TM to actin in the presence of Ca. The significant increase in affinity of the exon 9 chimeras, compared to TM9d, shows that both parts of exon 9a contribute, but that the effect is greatest with all 27 exon 9a residues. If only the first 18 residues of exon 9a were necessary for this strong (100-fold) promotion of binding, we would expect Tn in the presence of Ca to increase the actin affinity of TM9a/9d by more than the observed 4-fold effect. Although TM9a and TM9a/9d bind to TnT with similar affinity when measured using a solid phase affinity assay (BIAcore, Pharmacia) (^3)the ternary Tn complex in the presence of Ca only promotes the actin affinity of TM9a. Therefore, the first 18 residues encoded by exon 9a are sufficient for TnT binding, whereas both parts are necessary for promotion of TM binding to the regulated thin filament in the presence of Ca.

Another possible interpretation of the failure of Tn to greatly increase the actin affinity of TM9d or TM9a/9d in the presence of Ca is that they bind well in the absence of troponin and the effect of Tn is not additive. We do not favor this interpretation as previous studies have shown that the effect of Tn on striated muscle (acetylated) alpha-TM, which binds well in the absence of Tn, is additive, greatly increasing the affinity in the presence of Ca(16, 37) .

Exon 9a encodes what has long been recognized as the Ca-insensitive binding site of Tn for TM(17) , and our results support this model. Our finding that Tn in the presence of Ca does not promote actin binding of TM9a/ is consistent with previous analyses of carboxypeptidase-treated TM from which the last 11 residues were removed(42, 46) . Removal of the last 11 residues of TM weakens the binding of the NH(2)-terminal region of TnT (residues 1-158) to TM(17, 47, 48) , indicating the involvement of the entire COOH-terminal region of TM for Tn binding in the presence of Ca.

TM9d/ did not bind measurably to actin with Tn in the absence of Ca. We found this surprising since a reconstituted Tn complex with residues 159-259 of TnT binds to a TM affinity column (49) and promotes the actin binding of carboxypeptidase-treated striated alpha-TM in the absence of Ca(48) . The complex of TnC, TnI, and residues 159-259 of TnT is known to bind to TM in the region of Cys-190, referred to as the Ca-sensitive binding site, and has not been considered to bind to the extreme COOH terminus of TM(17, 48, 49, 50, 51) . The different affinities of TM9a/ and TM9d/ (which have the same sequence in the Cys-190 region) for regulated actin in the absence of Ca presumably reflect the differences in Tn affinity for the COOH terminus.

The properties of TM9d/ are comparable to TM with larger COOH-terminal deletions. A fusion striated alpha-TM lacking the last 39 residues does not bind to actin with Tn in the presence or absence of Ca(52) . Neither striated nor smooth beta-TM with the last 27 residues deleted bound well to actin(45) . Striated beta-TM with the last 31 residues deleted does not bind to actin with Tn in the presence of Ca, although it retains limited ability to regulate the actomyosin S1 ATPase(23) . The loss of function of TM9d/ is as great as that observed when the first nine residues are deleted from striated alpha-TM(53) . Troponin T extends across the overlap region and binds to the NH(2) terminus of the next TM on the thin filament(15, 54) . We suggest that the 27 COOH-terminal residues encoded by exon 9, the first 18 in particular, together with the extreme NH(2) terminus of TM are critical for striated TM-Tn assembly on the actin filament. In our analysis, it is not possible to distinguish between altered binding of TM directly to actin and altered end-to-end TM interactions on the thin filament. All unacetylated TMs polymerize poorly, even with Tn(19, 20) .

Comparing the sequences encoded by the first 18 codons of exons 9a and 9d (Fig. 2B), there are only three identities and four conservative changes; the striated form is slightly more acidic. In the absence of high resolution structural information, the sequences do not contribute to our understanding of the functional differences between the TM isoforms.

The ability of the TM exon 9 variants to regulate the actomyosin ATPase with Tn was insensitive to the observed differences in actin affinity. This is not surprising since at the concentrations used in the assays the thin filament would be fully saturated. Only TM9d/ was unable to regulate, an expected result since it did not bind to the thin filament. A more sensitive assay would be the cooperative activation of the thin filament by myosin S1, the extent of which is TM isoform specific(55, 56) . Unfortunately, unacetylated 284-residue TMs do not exhibit this activation(19, 23) , and in the presence of Tn and Ca, the activation of the acto-S1 ATPase is similar with acetylated striated alpha-TM, unacetylated striated alpha-TM, and unacetylated smooth alpha-TM(19, 53) . While we anticipate the COOH-terminal differences will contribute significantly to cooperative regulation by myosin S1, investigation of this TM function will require acetylated TMs produced in insect Sf9 cells(21) , the subject of future experiments.

Our results have given us insight into the structural requirements of TM for thin filament assembly and regulatory function. We now understand the functional significance of the striated-specific COOH terminus encoded by exon 9a in terms of a long-standing model of regulation. The binding of Tn in the Cys-190 region of TM is known to be stronger in the absence than in the presence of Ca, but in both conditions it is weaker than the binding of TnT to the COOH-terminal region of TM(47, 57) . Calcium binding to TnC weakens the binding of TnI and TnT to actin-TM in the region of Cys-190 of TM, and TnT is required in the Tn complex for it to remain associated with the thin filament (58-61; reviewed in (9) and (11) ). Troponin T, as well as the ternary Tn complex, greatly increases the affinity of TM for actin, a consequence of tight association between TnT and TM, as well as TnT and actin(62) . The work presented here indicates that the striated-specific TM COOH terminus, especially residues 258-275, is designed for strong, isoform-specific interaction of Tn (presumably TnT) with TM on the actin filament both in the presence and absence of Ca. Optimizing this interaction is important for thin filament assembly, inhibition of the actomyosin ATPase in the absence of Ca, and full activation in the presence of Ca.


FOOTNOTES

*
This work was supported in part by Grant HL35726 from the National Institutes of Health and 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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a predoctoral fellowship from the American Heart Association, NJ Affiliate. To whom correspondence should be addressed: Dept. of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 908-235-4528; Fax: 908-235-4029; :Hammell{at}mbcl.rutgers.edu.

(^1)
The abbreviations used are: TM, tropomyosin; Tn, troponin; DTT, dithiothreitol.

(^2)
Y. An and S. E. Hitchcock-DeGregori, unpublished results.

(^3)
R. L. Hammell and S. E. Hitchcock-DeGregori, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Norma Greenfield for discussions and CD analysis of the tropomyosin variants. We also thank Dr. Karen Ball and Kelley Nicholson for discussions and helpful comments on the manuscript.


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