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Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635
Correspondence: Address reprint requests to Thomas Palm, Tel.: 732-235-4528; Fax: 732-235-4029; E-mail: palmth{at}umdnj.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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-tropomyosin isoforms, and a troponin T fragment that binds to the tropomyosin overlap, to form complexes was analyzed using circular dichroism spectroscopy. Analysis of N-terminal extensions (N-acetylation, Gly, AlaSer) showed that to form an overlap complex between the N-terminus and the C-terminus requires that the N-terminus be able to form a coiled coil. Formation of a ternary complex with the troponin T fragment, however, effectively takes place only when the overlap complex sequences are those found in striated muscle tropomyosins. Striated muscle tropomyosins with N-terminal modifications formed ternary complexes with troponin T that varied in affinity in the order: N-acetylated > Gly > AlaSer > unacetylated. The circular dichroism results were corroborated by native gel electrophoresis, and the ability of the troponin T fragment to promote binding of full-length tropomyosins to filamentous actin. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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-helical protein that forms a two-stranded coiled coil. Tropomyosin molecules overlap at their ends by 
8–11 amino acids (McLachlan and Stewart, 1975
) to form long chains along the grooves of the actin filament. This way TM confers structural stability to the actin filament and modulates its function (reviewed in Perry, 2001). In eukaryotic cells TM exists in a large number of isoforms which are expressed by alternative promoters and alternative splicing of multiple genes (Lin et al., 1997).
Two major classes of tropomyosins in higher eukaryotes are the long, 284-residue tropomyosins expressed in muscle and nonmuscle cells, and the short 247-residue tropomyosins found in nonmuscle cells, that differ at the N-terminus (Lin et al., 1997). In the short tropomyosin isoforms, which span the length of six actin monomers in the filament, exon 1b (residues 1–44) replaces exon 1a and 2 (residues 1–80) expressed in the long isoforms, which span the length of seven actin monomers. Additional diversity results from alternative splicing of the other coding exons, including exon 9, which encodes the C-terminal 26 amino acids.
Studies of many different tropomyosin isoforms, both wild-type and recombinant forms, as well as nonnatural chimerae, have established that the N- and C-terminal regions of tropomyosin, and modifications to those sequences, alter tropomyosin binding to actin and troponin as well as the cooperative interaction with myosin on the actin filament (Mak and Smillie, 1981; Dabrowska et al., 1983; Heeley et al., 1987; Cho et al., 1990; Cho and Hitchcock-DeGregori, 1991; Willadsen et al., 1992; Butters et al., 1993; Novy et al., 1993a,b; Urbancikova and Hitchcock-DeGregori, 1994; Hammell and Hitchcock-DeGregori, 1996; Pittenger et al., 1995; Moraczewska et al., 1999; Sano et al., 2000; Cho, 2000). Of particular importance for striated muscle tropomyosin is N-acetylation of the initial methionine, a modification that stabilizes the coiled coil -helix and allows the N-terminus to be coiled coil to the end (Greenfield et al., 1994; Brown et al., 2001). In the absence of troponin, recombinant striated muscle tropomyosin expressed in Escherichia. coli, which is not acetylated, binds only weakly to actin. In the presence of troponin it binds well and can regulate the actomyosin ATPase. A fusion protein in which the N-terminal acetyl group is replaced by an 80-residue polypeptide binds well to actin, but the binding of troponin is Ca2+ sensitive and it does not confer effective calcium sensitivity on the actomyosin ATPase (Hitchcock-DeGregori and Heald, 1987; Heald and Hitchcock-DeGregori, 1988). The dipeptide AlaSer fused to the N-terminus of tropomyosin seems sufficient to replace the function of the N-terminal acetyl group in that it restores actin binding, head-to-tail polymerization, and the ability to inhibit the ATPase (Monteiro et al., 1994; Maytum et al., 2000).
The exon 9-specified C-terminus is also a major functional determinant. Replacement of the striated muscle specific exon 9a encoded C-terminus with that encoded by exon 9d, expressed in smooth and nonmuscle cells, allows unacetylated muscle tropomyosin to bind well to actin (Cho and Hitchcock-DeGregori, 1991). The higher affinity is attributable to the C-terminal nine amino acids encoded by exon 9d (Hammell and Hitchcock-DeGregori, 1996), in particular Gln276 and Thr 277 (Cho, 2000). However, the striated muscle specific exon 9a is required for binding to troponin T and for troponin to promote the binding of tropomyosin to actin (Cho and Hitchcock-DeGregori, 1991; Hammell and Hitchcock-DeGregori, 1996, 1997).
Our aim in the present work was to understand the isoform and structural specificity of the end-to-end association of tropomyosin molecules to form an overlap complex and to form a ternary complex with troponin T. For these studies we have used a model system that allows us to study the interaction of tropomyosin ends directly using N- and C-terminal model peptides, and a soluble troponin T fragment that binds to the C-terminus of tropomyosin (Palm et al., 2001). Model peptides have proven useful to study the structure and function of tropomyosin (Greenfield et al., 1994; Holtzer et al., 1995; Greenfield et al., 1998; Emerson Holtzer et al., 2001; Greenfield et al., 2001; Greenfield and Fowler, 2002). Here, with analysis of model peptides using circular dichroism we have been able to measure the affinity of the N-terminus for the C-terminus in forming an overlap complex, the conformational change that takes place upon binding, and the effect of N-terminal and C-terminal modifications on complex formation. Furthermore, the specificity for binding to troponin T to form a ternary overlap complex has been investigated, and correlated with the ability of troponin and the troponin T to promote binding of full-length tropomyosin to actin. The results give insight into the structural requirements and sequence specificity for formation of functional complexes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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), we selected the best expressed codons in E. coli using the software DNA* (DNASTAR, Inc., Madison WI). The DNA was cloned into pET11-HTb (Greenfield et al., 2002) between NdeI and KpnI, and expressed in E. coli BL21(DE3) (Studier et al., 1990). TM9a251–284 was isolated and oxidized as previously described (Greenfield et al., 2002). The typical yield of oxidized lyophilized peptide was 0.5 mg/l of culture medium.
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-TM with the sequence encoded by exon 9a with a Lys replacing the native Asn at position 279; TM9d246–284: last 39 residues of rat -TM with the sequence encoded by exon 9d) and N-terminal peptides (N-acetylated AcTM1aZip, AlaSerTM1aZip, unacetylated TM1aZip, and AcTM1bZip, Table 1) of tropomyosin were commercially synthesized, purified to >95% purity by reverse phase HPLC and analyzed by mass spectrometry (SynPep, Dublin, CA).
Recombinant tropomyosin and troponin T peptides
The N-terminal TM model peptide GlyTM1aZip was prepared as published earlier (Greenfield et al., 2002). The human cardiac troponin T fragment hcTnT70–170 was expressed in E. coli and purified as previously described (Palm et al., 2001).
Recombinant tropomyosin isoforms and chicken skeletal -tropomyosin
DNA constructs and protein expression for the rat -tropomyosin isoforms 1a9a, 1a9d, 1b9a, and 1b9d have been previously described (Cho and Hitchcock-DeGregori, 1991; Hammell and Hitchcock-DeGregori, 1996; Moraczewska et al., 1999). The cDNA for AlaSer-tropomyosin was made from rat striated TM cDNA in a pET11d vector using the oligonucleotide primers 5'-CATATGGCTTCTATGGACGCCATCAAGAAGAAGCGCC-3' and 5'-TATATCTCCTTCTTAAAGTTAAACAAAATTATTTC-3' and standard PCR techniques. The product contained a novel NdeI site to facilitate selection of positive clones. AlaSer-TM was expressed in E. coli BL21(DE3). All recombinant tropomyosin isoforms were purified as described previously (Hitchcock-DeGregori and Heald, 1987; Hammell and Hitchcock-DeGregori, 1996; Moraczewska et al., 1999). The N-termini are unacetylated. Acetylated striated muscle -tropomyosin was purified from the isoelectric precipitate from a troponin preparation from chicken pectoral muscle (Hitchcock-DeGregori et al., 1985).
Circular dichroism studies
Circular dichroism (CD) data were collected on an Aviv model 62D spectropolarimeter (Aviv, Lakewood, NJ) equipped with a five-sample thermal-equilibration chamber as previously described (Greenfield and Hitchcock-DeGregori, 1995). N-terminal TM-peptides were measured either alone, or in equimolar mixtures with C-terminal TM model peptides (overlap complex), or with C-terminal TM model peptides and hcTnT70–170 (ternary complex).
Data were collected at a concentration of 10 µM for each peptide in 10 mM potassium phosphate, pH = 6.5. Circular dichroism spectra were measured at 0°C and the -helical content of the TnT peptides was calculated from ellipticity data using the neural network program, CDNN (Böhm et al., 1992). Thermal stability measurements were performed by following the ellipticity at 222 nm as a function of temperature between 0°C and 70°C. Unless mentioned otherwise, the midpoints of the thermal transitions (Tm) were determined from fits of the melting curves to the Gibbs-Helmholtz equation for a single transition. The enthalpy (
H) and entropy (S) of folding for the tropomyosin and troponin T peptides were determined from the change in ellipticity as previously described (Greenfield et al., 1998). To estimate H and S for the binary tropomyosin overlap complexes, the melting curves were fit to the Gibbs-Helmholtz equation for a single transition, assuming that one mole of the complex dissociates into one mole of each of the components. For the ternary peptide complexes of troponin T and the tropomyosin overlap, the enthalpy and entropy of folding were determined by fitting the CD of the complex as a function of temperature to the linear van't Hoff equation. Apparent binding constants were estimated from the free energy of folding (G) of the binary and ternary peptide complexes at 20°C. The free energy of folding was calculated from H and S using the Gibbs equation, G = H - TS, in which T is the absolute temperature and H and S were assumed to be independent of temperature. Using the assumption that the entire difference in G between the complexes and their components (G) was due to binding, the apparent dissociation constant was estimated using the equation Kd(app) = 1/exp(-G/nRT). These estimates depend on the fact that the complexes unfolded in two-state reversible cooperative transitions. This was the case for all complexes that formed except the ternary complex that involved unacetylated TM1aZip. The apparent dissociation constants reported here for the ternary complexes are the dissociation constants of the troponin T peptide for the tropomyosin overlap. The apparent dissociation constant for the whole ternary complex would be the product of the dissociation constant of the TM overlap complex and the dissociation constant of the troponin T peptide for the tropomyosin overlap complex.
Native gel electrophoresis
Samples of ternary complexes of N-terminal TM model peptides with TM9a251–284 and hcTnT70–170 for native gel electrophoresis were prepared at a concentration of 10 µM in 10 mM potassium phosphate, pH = 6.5, 10% glycerol. Ten µl samples were run at 4°C on 10% acrylamide, 10% glycerol gels in 20 mM Tris, pH = 8.8, 120 mM glycine (Katayama and Nozaki, 1982). Gel bands were quantitated using a Molecular Dynamics 300A computing densitometer (Molecular Dynamics, Sunnyvale, CA).
Tropomyosin binding to F-actin
The effect of hcTnT70–170 on the affinity of TM isoforms for actin was measured by co-sedimentation. Tropomyosin was combined with hcTnT70–170 in a 1:1.2 molar ratio in 100 or 300 mM NaCl, 2 mM MgCl2, 0.5 mM DTT, 10 mM Tris, pH 7.5 and centrifuged at 60,000 rpm, 20°C, for 25 min in a Beckman TLA-100 rotor to remove precipitated protein. F-actin (5 µM final concentration) was combined with the TM and hcTnT70–170 mixture (0–6 µM final concentration) and centrifuged as before. The actin and TM were quantitated in SDS polyacrylamide gels of the pellets and supernatants using a Molecular Dynamics 300A computing densitometer. The data were fit to the Hill equation to determine the affinity of TM for actin:
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is the observed TM/actin density ratio at [TM], n is the maximal TM/actin density ratio, Kdapp is the apparent equilibrium constant at which 50% of TM is bound to actin, and H is the Hill coefficient.
General methods
Skeletal muscle actin was prepared from chicken pectoral muscle acetone powder (Hitchcock-DeGregori et al., 1982). SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (1970). Unless mentioned otherwise, the concentration of proteins and peptides was determined with the micro biuret method (Goa, 1953).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). The tropomyosin C-terminus is a 37-residue peptide, TM9a251–284, consisting of residues 251–284 of rat striated -tropomyosin with the N-terminal extension Gly-Cys-Gly to allow oxidative cross-linking via disulfide bond formation (Table 1). The recombinant peptide includes the entire region encoded by striated muscle specific exon 9a (residues 258–284) plus seven amino acids encoded by the constitutively expressed exon 8. To compare the function of the striated muscle-specific exon 9a C-terminus with the smooth/nonmuscle tropomyosin exon 9d-encoded C-terminus we used synthetic model peptides with the last 39 amino acids (TM9a246–284N279K, TM9d246–284). The TM9a246–284N279K peptide contains a functionally neutral Asn279Lys mutation that increases the stability of the peptide (Palm et al., 2001).
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, 2001). In both, the C-terminal 18 residues are the last 18 C-terminal residues of the GCN4 transcription factor leucine zipper (Landschulz et al., 1988), included to promote and stabilize formation of a parallel two-chained coiled coil. For the N-terminus found in long TMs (all muscle tropomyosins as well as many nonmuscle isoforms), the first 14 amino acids are residues 1–14 encoded by rat -tropomyosin exon 1a (TM1aZip). Modifications of the N-terminal methionine will be discussed below. The alternative N-terminal peptide for short tropomyosin isoforms, TM1bZip, includes residues 1–19 encoded by rat -tropomyosin exon 1b, where the N-terminal Ala is acetylated (in a synthetic peptide, AcTM1bZip). This peptide contains 14 residues of exon 1b that are homologous to exon 1a plus a native five amino acid N-terminal extension (Table 1; Lewis et al., 1983; Greenfield et al., 2001). The hcTnT70–170 is a recombinant fragment of human cardiac TnT that binds well to the C-terminus of striated muscle -tropomyosin and stabilizes the overlap complex with the N-terminus (Palm et al., 2001).
Isoform specificity of formation of binary and ternary overlap complexes
Our work with full-length recombinant tropomyosins established the critical importance for the alternatively expressed N- and C-terminal sequences in determining actin affinity and binding to troponin T (Cho and Hitchcock-DeGregori, 1991; Hammell and Hitchcock-DeGregori, 1996, 1997; Moraczewska et al., 1999). To study the specificity of the N- and C-terminal overlap formation we used the 39-residue, uncross-linked model peptides, TM9a246–284N279K and TM9d246–284.
Circular dichroism in 10 mM phosphate was used to determine the secondary structure and thermal stability of tropomyosin model peptides. Initial experiments were also performed at physiological salt concentrations. These experiments showed essentially the same results as the experiments without salt but the total effect was smaller. Therefore, only the experiments without salt are discussed here. The increase in -helix and helical stability, as well as an increase in the cooperativity of unfolding when the peptides were combined, were used as measures of complex formation. Apparent dissociation constants of peptide complexes were estimated from van't Hoff enthalpies. Recently several papers have shown that binding constants determined from van't Hoff enthalpies of folding are equivalent to those determined from direct measurements of the enthalpies of binding determined by isothermal calorimetry (Horn et al., 2001, 2002). Also, in a similar study of tropomyosin binding to tropomodulin fragments we found that the apparent dissociation constants determined from the denaturation curves are within experimental error to those determined by direct titration (Greenfield and Fowler, 2002).
Even though the 39-residue C-terminal peptides are less helical and less stable than the cross-linked peptide TM9a251–284 (see below), they served to illustrate isoform specific complex formation (Table 2). Both C-terminal peptides formed more stable complexes with AcTM1bZip (the N-acetylated peptide, or with GlyTM1bZip where Gly replaces the acetyl group, unpublished results) than with AcTM1aZip. However, hcTnT70–170 formed a convincingly stable ternary complex only with AcTM1aZip and TM9a246–284N279K, resulting in a small increase in Tm, a marked increase in negative ellipticity (Table 2), and an increase in the enthalpy and entropy of unfolding, as a function of temperature as reflected by the increased cooperativity (i.e., steepness) of the melting curves (data not shown).
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; Urbancikova and Hitchcock-DeGregori, 1994; Monteiro et al., 1994; Maytum et al., 2000). Here we investigated the structural requirements of the tropomyosin N-terminus to form binary and ternary overlap complexes. For these studies we used a shorter C-terminal peptide, TM9a251–284, that contains a cysteine. The increased stability of the cross-linked TM9a251–284 peptide allowed detection of stable binary and ternary complexes, that was difficult with the uncross-linked, TM9a246–284N279K peptide (Table 2).
Conformational stability of binary and ternary overlap complexes measured using circular dichroism
AcTM1aZip and TM9a251–284 formed a stable binary overlap complex, as reflected in greater cooperativity of unfolding as well as a slight increase of -helix and an increase in Tm of 9.4°C (Tm, the midpoint of the thermal transition of the mixture versus the sum of components, Fig. 2, Table 3). Addition of an equimolar amount of hcTnT70–170 to the binary overlap complex resulted in a further Tm of 7.4°C, indicating the formation of a stable ternary complex of the striated muscle overlap with the N-terminal part of TnT (Table 4).
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, 2001; Brown et al., 2001), we made three N-terminal modifications of AcTM1aZip, t1he wild-type form: unacetylated (with a free amino group on Met1), Gly preceding Met1, and AlaSer preceding Met1. Both unacetylated and GlyTM1aZip are unstable compared to AcTM1aZip with Tm temperatures of less than 10°C, whereas AlaSerTM1aZip is intermediate (Table 3). Modification of Met1 also affects complex formation with the C-terminus, as measured by an increase in Tm and cooperativity of thermal denaturation. The Gly modification is almost as effective as N-acetylation in binary complex formation, whereas AlaSerTM1aZip and AcTM1bZip form even stronger complexes (Fig. 2, Table 3). However, the stability and cooperativity of unfolding of the mixture of unacetylated TM1aZip withTM9a251–284 are only marginally greater than the sum of the components.
The N-terminal structure also has a major influence on the formation of a ternary complex with hcTnT70–170 (Fig. 2, Table 4). The Tm for the ternary complex with GlyTM1aZip complex was close to that of AcTM1aZip, accompanied by an increased enthalpy and entropy of unfolding as reflected by the increased cooperativity (i.e., steepness) of the melting curves (Fig. 4). The Tm of the ternary complex with unacetylated TM1aZip complex was smaller, and the analysis was complicated by the presence of two transitions indicating that the ternary complex dissociates before the TnT fragment unfolds. In contrast, the stable binary complex formed with AlaSerTM1aZip was considerably less effective in forming a ternary complex with hcTnT70–170 than AcTM1aZip or GlyTM1aZip, having a Tm for the ternary complex of only 2°C. No obvious change was seen with AcTM1bZip.
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In general, the results agree with the CD results, even though the gel was run at a higher pH. Ternary complexes were observed for AcTM1aZip, GlyTM1aZip, and AlaSerTM1aZip. Although the increase in thermal stability (Tm) for the ternary complexes with AcTM1aZip or GlyTM1aZip was similar, and higher than with AlaSerTM1aZip, the overall stability of the AcTM1aZip ternary complex is higher than both in native gels. No ternary complexes were observed for AcTM1bZip and unacetylated TM1aZip indicating that these complexes either did not form in the conditions of the native gel, or were not sufficiently stable to remain intact during gel electrophoresis. The only binary complex stable enough to be seen on the native gel was that with AcTM1bZip. The binary complex with AlaSerTM1aZip is of similar stability according to the CD data but was not detected on the native gel, probably because of the difference in pH of the buffers used for the CD and electrophoresis studies. Although the sequences are homologous, the 1b peptide has a region where RRK replaces KKK in the 1a peptide. The 1b peptide might be expected to be less affected by the high pH of the electrophoresis buffer (pH 8.8) than the 1a peptide because the pKa of arginine is higher than that of lysine.
Promotion of binding of N-terminal variants of tropomyosin to actin by hcTnT70–170
N-terminal modifications such as N-acetylation, AlaSer, or the exon 1b N-terminus increase the end-to-end affinity of striated muscle tropomyosin compared to unacetylated TM (Heald and Hitchcock-DeGregori, 1988; Willadsen et al., 1992; Urbancikova and Hitchcock-DeGregori, 1994; Monteiro et al., 1994; Moraczewska et al., 1999; Maytum et al., 2000). To correlate the specificity of the N-terminal tropomyosin structure in formation of a ternary overlap complex with actin affinity we measured the ability of hcTnT70–170 to promote the binding of four N-terminal tropomyosin variants with an exon 9a-encoded C-terminus, and one with an exon 9d-encoded C-terminus to filamentous actin (Fig. 4, Table 5). The sequences are identical except at the ends. The N-terminal and C-terminal sequences found in striated muscle tropomyosin are both required for TnT to form a strong ternary overlap complex and to increase the affinity of tropomyosin for actin. The hcTnT70–170 fragment was most effective in increasing the affinity of both acetylated (Fig. 4 A) and unacetylated (Fig. 4 B) striated muscle tropomyosin (with exons 1a and 9a) for actin 10- to 15-fold (Table 5), in agreement with previously published results (Wegner and Walsh, 1981; Heald and Hitchcock-DeGregori, 1988; Hinkle et al., 1999). The binding of AlaSerTM (with exons 1a and 9a) was enhanced only threefold by hcTnT70–170, less than the acetylated and unacetylated forms (Fig. 4A, Table 5). In these studies, the AlaSer1a9aTM was more similar to TM1b9a, consistent with the CD results using model peptides, even though it has both muscle tropomyosin exons. The hcTnT70–170 fragment had the smallest influence on tropomyosins with exon 1b, or exon 9d (Fig. 4, A and B; Table 5), as in the CD and gel electrophoresis experiments reported above, and in experiments using native troponin complex (Cho and Hitchcock-DeGregori, 1991; Hammell and Hitchcock-DeGregori, 1996; Moraczewska et al., 1999).
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DISCUSSION |
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The mixtures of all of the N-terminal peptides with both the 9a and 9d C-terminal peptides, with the exception of unacetylated TM1aZip, have greatly increased stabilities (>8°C) compared to the sum of the unmixed components. The published structures of the N-terminus of tropomyosin give insight into the meaning of these results. In AcTM1aZip and AcTM1bZip, two peptides that form stable binary complexes, the regions proposed to participate in the overlap complex are -helical coiled coils (residues 1–11 and the homologous region in TM1bZip, residues 6–16; Greenfield et al., 1998, 2001). When fully folded, AlaSerTM1aZip and GlyTM1aZip have the same ellipticity at 222 nm as AcTM1aZip (from extrapolation of unfolding curves or when measured at higher peptide concentrations, data not shown) showing that they form essentially the same structure as the acetylated peptide. In contrast, the first two residues of unacetylated 1a tropomyosins are not helical and are not part of the coiled coil domain (Brown et al., 2001). This is reflected in the ellipticity of the fully folded unacetylated peptide at 222 nm, which is 30% lower than that of the acetylated peptide (data not shown). The unacetylated peptide is also less stable than the acetylated peptide. In summary, we suggest that the entire region homologous to residues 1–11 in AcTM1aZip must be able to form a fully folded coiled coil -helix to form a strong overlap complex with the C-terminus.
It was impossible to determine the relative affinities of the N-terminal tropomyosin peptides for the TM9a246–284N279K and TM9d246–284 peptides because of the very low stability of the 9d peptide. However, it is clear that all four complexes can be readily formed since in each case there are increases in the ellipticities of the mixtures compared to the sum of the ellipticities of the unmixed components.
Formation of a ternary complex with the N-terminus of troponin T, on the other hand, depends on the isoform specificity of both the N-terminal and C-terminal parts of the overlap complex. Troponin T is only expressed in striated muscle. The specificity is most clearly illustrated by the requirement for both striated tropomyosin exon 1a and exon 9a-encoded termini for strong ternary complex formation, as is required for troponin to promote binding of tropomyosin to actin (Cho and Hitchcock-DeGregori, 1991; Hammell and Hitchcock-DeGregori, 1996; Moraczewska et al., 1999). The overlap complex containing the N-terminus encoded by exon 1a has almost a 20-fold higher affinity for the TnT peptide than the complex containing the 1b N-terminus. Adding the AlaSer extension to the 1a N-terminus decreases the binding affinity of the overlap complex for the TnT peptide more than tenfold, suggesting that the AlaSer extension is mimicking the N-terminus of the 1b tropomyosins, rather than the acetyl group of native striated muscle TM1a. This suggestion is supported by the fact that the N-terminus of 1b tropomyosins is homologous to the N-terminus of 1a tropomyosins but has a native five amino acid N-terminal extension AlaGlySerSerSer which resembles AlaSer.
Although AlaSerTM1a and TM1b are impaired in binding the TnT peptide, the modifications increase actin affinity in the absence of TnT, as previously reported (Monteiro et al., 1994; Moraczewska et al., 1999). These results are consistent with results of Maytum et al. (2000; 2001) who find little difference in the actin affinities of yeast tropomyosin with various N-terminal extensions.
A full understanding of the structural requirements and specificity of the overlap complex and ternary complex with troponin T awaits atomic resolution structures. Toward that goal, we recently reported that binding of GlyTM1aZip to a related TM9a peptide, strongly perturbs the crosspeaks of residues 274–284 in its 1H-15N HSQC spectrum (Greenfield et al., 2002). In addition, the binding of the TM9a peptide to GlyTM1aZip perturbs the crosspeaks arising from residues 1–8 and 11–12 (unpublished results). Together the results suggest that the first and last 11 residues interact in the overlap complex. We have previously proposed models where the overlap complex of the TM1a and TM9a ends interact with residues 92 to 110 of troponin T to form a five-helix coiled coil or helix bundle. The five-residue nonhelical extension of the 1b tropomyosins and the AlaSer extension could inhibit binding of the TnT peptide by steric hindrance.
An alternate explanation is that the binary complexes of the TM9a peptide with the AlaSerTM1aZip and TM1bZip peptides, which are considerably more stable than the AcTM1aZip complex, are too stable for TnT to bind. A mutation, Q263L, that increases the stability of the TM9a peptide decreases the ability of its overlap complex to bind the hcTnT70–170 fragment (Greenfield et al., 2002), suggesting that conformational flexibility is necessary for tropomyosin to bind TnT. Similarly, disease causing mutations that increase the -helical stability of the TnT peptide reduce its affinity for tropomyosin and the overlap complex (Palm et al., 2001).
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ACKNOWLEDGEMENTS |
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This research was supported by National Institutes of Health grant RO1 GM-36326 to S.E.H.-D. and N.J.G. and a shared instrumentation grant 1 S10 RR 16705-01 to the circular dichroism facility at UMDNJ.
Submitted on September 18, 2002; accepted for publication December 26, 2002.
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REFERENCES |
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Brown, J. H., K. H. Kim, G. Jun, N. J. Greenfield, R. Dominguez, N. Volkmann, S. E. Hitchcock-DeGregori, and C. Cohen. 2001. Deciphering the design of the tropomyosin molecule. Proc. Natl. Acad. Sci. USA. 98:8496–8501.
Butters, C. A., K. A. Willadsen, and L. S. Tobacman. 1993. Cooperative interactions between adjacent troponin-tropomyosin complexes may be transmitted through the actin filament. J. Biol. Chem. 268:15565–15570.
Cho, Y. J. 2000. The Carboxyl Terminal Amino Acid Residues Glutamine276-Threonine277 Are Important for Actin Affinity of the Unacetylated Smooth -Tropomyosin. J. Biochem. Mol. Biol. 33:531–536.
Cho, Y. J., and S. E. Hitchcock-DeGregori. 1991. Relationship between alternatively spliced exons and functional domains in tropomyosin. Proc. Natl. Acad. Sci. USA. 88:10153–10157.
Cho, Y. J., J. Liu, and S. E. Hitchcock-DeGregori. 1990. The amino terminus of muscle tropomyosin is a major determinant for function. J. Biol. Chem. 265:538–545.
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), in complex with TM9a251–284 (
), and in complex with TM9a251–284 and hcTnT70–170 (
). Solid lines represent fits to the mixtures, and dotted lines represent fits to the sum of the components. Thermal transitions of TM9a251–284 (
) and hcTnT70–170 (
) are shown in panel F.
