Prediction of a triple-stranded coiled-coil region in tropomyosin-troponin T complex

Recent studies (Ohtsuki, 1979, 1980) have shown that troponin T (tropomysin binding component of troponin complex) is a rod-shaped molecule of approximately 9 nm in length and associated with filamentous tropomyosin. The region of residues 90 to 148 of troponin T, which has been confirmed as a main portion of helix-rich fragment which strongly binds to tropomyosin (Jackson, Amphlett & Perry, 1975, Pearlstone & Smillie, 1977), was predicted as a long stretch of a -helix by the method of secondary structure prediction (Nagano, 1977, Nagano et al., 1980). This paper deals with the mechanism of the specific binding explored by the extension of the method of schematic representation of helical interactions developed by McLachlan & Stewart (1976a) and, also, the method of scoring interactions of the staggered structures of collagen triplestranded coiled-coils developed by Hulmes et al. (1973). One of the most feasible structures of the specific binding complex was obtained as a triple-stranded coiled-coil made between a tropomyosin coiled-coil and the helical region of the specific binding fragment of troponin T, and confirmed by both LabQuip type and computer model building techniques. The techniques developed in the present work will be useful in elucidating the regulating mechanism of muscle contraction in its atomic details. The procedures and the steps for restricting the number of possible structures are outlined in Table 4.     

Disease-causing mutations in cardiac troponin T: identification of a critical tropomyosin-binding region

Fifteen percent of the mutations causing familial hypertrophic cardiomyopathy are in the troponin T gene. Most mutations are clustered between residues 79 and 179, a region known to bind to tropomyosin at the C-terminus near the complex between the N- and C-termini. Nine mutations were introduced into a troponin T fragment, Gly-hcTnT(70-170), that is soluble, alpha-helical, binds to tropomyosin, promotes the binding of tropomyosin to actin, and stabilizes an overlap complex of N-terminal and C-terminal tropomyosin peptides. Mutations between residues 92 and 110 (Arg92Leu, Arg92Gln, Arg92Trp, Arg94Leu, Ala104Val, and Phe110Ile) impair tropomyosin-dependent functions of troponin T. Except for Ala104Val, these mutants bound less strongly to a tropomyosin affinity column and were less able to stabilize the TM overlap complex, effects that were correlated with increased stability of the troponin T, measured using circular dichroism. All were less effective in promoting the binding of tropomyosin to actin. Mutations within residues 92-110 may cause disease because of altered interaction with tropomyosin at the overlap region, critical for cooperative actin binding and regulatory function. A model for a five-chained coiled-coil for troponin T in the tropomyosin overlap complex is presented. Mutations outside the region (Ile79Asn, Delta 160Glu, and Glu163Lys) functioned normally and must cause disease by another mechanism.     

The troponin binding region of tropomyosin. Evidence for a site near residues 197 to 127

Study of magnesium paracrystals has shown that the troponin binding region of tropomyosin is within about 30 Å of a dyad axis which lies close to Cys190 and Leu197. The region of α-tropomyosin between residues 197 and 217 has an exceptionally small number of negative charges, a significantly high concentration of uncharged polar groups, and a large hydrophobic surface. These features suggest that this is the main binding site for troponin T. A second weaker site for calcium-sensitive binding to troponin could exist on the opposite face of the symmetrical double helix.     

Mode of degradation of myofibrillar proteins by rabbit muscle cathepsin D

The mode of degradation of myofibrillar proteins by the action of highly purified rabbit muscle cathepsin D (EC 3.4.23.5) was studied using SDS-polyacrylamide gel electrophoresis. Cathepsin D optimally degraded myosin heavy chain, α-actinin, tropomyosin, troponin T and troponin I at around pH 3. It did not degrade actin or troponin C. Degradation of myosin heavy chain produced four major fragments of 155 000, 130 000, 110 000 and 90 000 daltons. Troponin T was hydrolyzed to 33 000-, and 20 000- and 11 000-dalton fragments. Troponin I was degraded into fragments of 13 000 and 11 000 daltons. Degradation of α-actinin and tropomyosin was not as rapid as that of mysoin and troponins T and I. Tropomyosin gave a fragment of 30 000 daltons, but α-actinin showed no distinct band of this fragment on gels.     

Chymotryptic subfragments of troponin T from rabbit skeletal muscle. Interaction with tropomyosin, troponin I and troponin C

The binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14 mM Tris/90 mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.     

Molecular polarity in tropomyosin-troponin T co-crystals.

New features of the structure and interactions of troponin T and tropomyosin have been revealed by electron microscopy of so-called double-diamond co-crystals. These co-crystals were formed using rabbit alpha2 tropomyosin complexed with troponin T from either skeletal or cardiac muscle, which have different lengths in the amino-terminal region, as well as a bacterially expressed skeletal muscle troponin T fragment of 190 residues that lacks the amino-terminal region. Differences in the images of the co-crystals have allowed us to establish the polarities of both the troponin T subunit and tropomyosin in the projected lattice. Moreover, in agreement with their sequences, the amino-terminal region of a bovine cardiac muscle troponin T isoform appears to be longer than that from the rabbit skeletal muscle troponin T isoform and to span more of the amino terminus of tropomyosin at the head-to-tail filament joints. Images of crystals tilted relative to the electron beam also reveal the supercoiling of the tropomyosin filaments in this lattice. Based on these results, a three-dimensional model of the double-diamond lattice has been constructed.     

Structure-function relationships in cardiac troponin T

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.     

Ca2+-induced rolling of tropomyosin in muscle thin filaments: the alpha- and beta-band hypothesis revisited

Tropomyosin is a filamentous coiled-coil protein directly involved in the regulation of the actomyosin interaction responsible for muscle contraction: it transmits the local calcium-induced conformational change in troponin to the helical array of myosin-binding sites on the surface of the actin filament. McLachlan and Stewart (McLachlan, A. D., and Stewart, M. (1976) J. Mol. Biol. 103, 271-298) proposed that the tropomyosin coiled-coil structure can be divided into 14 alternating 19- to 20-residue "alpha- and beta-bands," which could act as alternate 7-fold sets of sites for specific binding to actin in the different conformational states of the regulated thin filament. Here we present the first direct experimental evidence in support of the alpha- and beta-band hypothesis: we analyze the acrylamide quenching of the fluorescence of mutant tropomyosins containing 5-hydroxytryptophan residues at different positions along the coiled-coil structure under a variety of conditions (alone, complexed with actin, and complexed with actin and troponin with or without Ca(2+)). We show that fluorescent probes placed in the alpha-bands become less solvent-exposed in the absence of calcium, whereas those in the beta-bands become less solvent-exposed in the presence of calcium. A model in which the tropomyosin coiled-coil rolls across the actin surface in response to Ca(2+)-binding to troponin most easily explains these observations.     

Regulatory properties of recombinant tropomyosins containing 5-hydroxytryptophan: Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site.

We have introduced tryptophan codons at different positions of the chicken alpha-tropomyosin cDNA (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466) and employed a trp auxotrophic Escherichia coli strain to express the proteins in media containing either normal tryptophan, 5-hydroxytrptophan, or 7-azatryptophan. The fluorescence of these latter two tryptophan analogues is excitable at 312-315 nm at which the natural fluorescence of other thin filament proteins (actin, troponin) is not excited. The recombinant tropomyosins have tryptophans or analogues located at amino acid positions 90, 101, 111, 122, or 185 of the protein, all on the external surface of the tropomyosin coiled-coil (positions "c" or "f" of the hydrophobic heptad repeat). The first four mutations are located within the third actin-binding zone of tropomyosin, a region not expected to interact directly with troponin or with neighboring tropomyosin molecules in muscle thin filaments, while position 185 is located in a region that has been implicated in interactions with the globular domain of troponin. The fluorescence intensity of the mutant containing 5-hydroxytryptophan at position 122 (5OH122W) is sensitive to actin binding and sensitive to Ca2+-binding to thin filaments reconstituted with troponin. Assuming that the globular domain of troponin binds to a site between residues 150 and 190 of tropomyosin, the distance between the troponin-binding site and the fluorescent probes at position 122 can be estimated to be 4.2-10.2 nm. While X-ray diffraction and electron micrograph reconstitution studies have provided evidence of Ca2+-induced changes in tropomyosin's interactions in the thin filament, their resolution was not sufficient to distinguish between changes involving the whole tropomyosin molecule or only that region directly interacting with troponin. Here we provide a clear demonstration that Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site which is probably associated with a changed interaction with actin.     

The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin

Analysis of two recombinant variants of chicken striated muscle alpha-tropomyosin has shown that the structure of the amino terminus is crucial for most aspects of tropomyosin function: affinity to actin, promotion of binding to actin by troponin, and regulation of the actomyosin MgATPase. Initial characterization of variants expressed and isolated from Escherichia coli has been published (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Fusion tropomyosin contains 80 amino acids of a nonstructural influenza virus protein (NS1) on the amino terminus. Nonfusion tropomyosin is a variant because the amino-terminal methionine is not acetylated (unacetylated tropomyosin). The affinity of tropomyosin labeled at Cys190 with N-[14C]ethylmaleimide for actin was measured by cosedimentation in a Beckman Airfuge. Fusion tropomyosin binds to actin with an affinity slightly greater than that of chicken striated muscle alpha-tropomyosin (Kapp = 1-2 X 10(7) versus 0.5-1 X 10(7) M-1) and more strongly than unacetylated tropomyosin (Kapp = 3 X 10(5) M-1). Both variants bind cooperatively to actin. Troponin increases the affinity of unacetylated tropomyosin for actin (+Ca2+, Kapp = 6 X 10(6) M-1; +EGTA, Kapp = 2 X 10(7) M-1), but the affinity is still lower than that of muscle tropomyosin for actin in the presence of troponin (Kapp much greater than 10(8) M-1). Troponin has no effect on the affinity of fusion tropomyosin for actin indicating that binding of troponin T to the over-lap region of the adjacent tropomyosin, presumably sterically prevented by the fusion peptide in fusion tropomyosin, is required for troponin to promote the binding of tropomyosin to actin. The role of troponin T in regulation and the mechanisms of cooperative binding of tropomyosin to actin have been discussed in relation to this work.     

Competitive binding of the troponin T-specific pool of caldesmon antibodies and tropomyosin to skeletal troponin T and smooth muscle caldesmon.

The fraction of polyclonal caldesmon antibodies cross-reacting with rabbit skeletal troponin T are shown to compete with smooth muscle tropomyosin for caldesmon and troponin T, as revealed by ELISA method. The epitope recognized by these antibodies was also found in Mr 77 kDa non-muscle caldesmon. These results provide functional confirmation for the suggestion that the regions of amino acid sequence homology in caldesmon isoforms and troponin T belong to the tropomyosin binding sites.     

Cardiac tropomyosin crystals and their interactions with troponin subunits

Crystals and paracrystals of bovine cardiac tropomyosin and their mixtures with different combinations of troponin subunits were examined in the electron microscope after negative staining. Although the cardiac proteins gave most of the same crystalline and paracrystalline patterns as observed previously with skeletal muscle tropomyosin and troponin, two important differences were noted. Cardiac troponin T was incapable of forming hexagonal networks with either skeletal or cardiac muscle tropomyosins, while skeletal troponin T readily associated in this manner with tropomyosins from either tissue source. Also the characteristic paracrystalline pattern seen with skeletal muscle tropomyosin, troponin T and troponin C only in the presence of calcium was consistently obtained with mixtures of the corresponding cardiac components when calcium was absent.     

Troponin T core structure and the regulatory NH2-terminal variable region

The conserved central and COOH-terminal regions of troponin T (TnT) interact with troponin C, troponin I, and tropomyosin to regulate striated muscle contraction. Phylogenic data show that the NH2-terminal region has evolved as an addition to the conserved core structure of TnT. This NH2-terminal region does not bind other thin filament proteins, and its sequence is hypervariable between fiber type and developmental isoforms. Previous studies have demonstrated that NH2-terminal modifications alter the COOH-terminal conformation of TnT and thin filament Ca2+-activation, yet the functional core structure of TnT and the mechanism of NH2-terminal modulation are not well understood. To define the TnT core structure and investigate the regulatory role of the NH2-terminal variable region, we investigated two classes of model TnT molecules: (1) NH2-terminal truncated cardiac TnT and (2) chimera proteins consisting of an acidic or basic skeletal muscle TnT NH2-terminus spliced to the cardiac TnT core. Deletion of the TnT hypervariable NH2-terminus preserved binding to troponin I and tropomyosin and sustained cardiac muscle contraction in the heart of transgenic mice. Further deletion of the conserved central region diminished binding to tropomyosin. The reintroduction of differently charged NH2-terminal domains in the chimeric molecules produced long-range conformational changes in the central and COOH-terminal regions to alter troponin I and tropomyosin binding. Similar NH2-terminal charge effects are demonstrated in naturally occurring cardiac TnT isoforms, indicating a physiological significance. These results suggest that the hypervariable NH2-terminal region modulates the conformation and function of the TnT core structure to fine-tune muscle contractility.     

The structure of the carboxyl terminus of striated alpha-tropomyosin in solution reveals an unusual parallel arrangement of interacting alpha-helices

Coiled coils are well-known as oligomerization domains, but they are also important sites of protein-protein interactions. We determined the NMR solution structure and backbone (15)N relaxation rates of a disulfide cross-linked, two-chain, 37-residue polypeptide containing the 34 C-terminal residues of striated muscle alpha-tropomyosin, TM9a(251-284). The peptide binds to the N-terminal region of TM and to the tropomyosin-binding domain of the regulatory protein, troponin T. Comparison of the NMR solution structure of TM9a(251-284) with the X-ray structure of a related peptide [Li, Y., Mui, S., Brown, J. H., Strand, J., Reshetnikova, L., Tobacman, L. S., and Cohen, C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7378-7383] reveals significant differences. In solution, residues 253-269 (like most of the tropomyosin molecule) form a canonical coiled coil. Residues 270-279, however, are parallel, linear helices, novel for tropomyosin. The packing between the parallel helices results from unusual interface residues that are atypical for coiled coils. Y267 has poor packing at the coiled-coil interface and a lower R(2) relaxation rate than neighboring residues, suggesting there is conformational flexibility around this residue. The last five residues are nonhelical and flexible. The exposed surface presented by the parallel helices, and the flexibility around Y267 and the ends, may facilitate binding to troponin T and formation of complexes with the N-terminus of tropomyosin and actin. We propose that unusual packing and flexibility are general features of coiled-coil domains in proteins that are involved in intermolecular interactions.     

Synthesis and biological activity of an icosapeptide analog of the actomyosin ATPase inhibitory region of troponin I.

A 20-residue peptide analog of the actomyosin ATPase inhibitory region of rabbit skeletal troponin I (Tn-I) has been synthesized by the solid phase method. The analog exhibited biological activity similar to both Tn-I and a 21-residue cyanogen bromide fragment of Tn-I. At ionic strengths where the inhibition of the actomyosin ATPase due to tropomyosin alone is low, the synthetic peptide in the presence of tropomyosin inhibits 90% of the original ATPase activity. In the absence of tropomyosin, the inhibition due to the peptide is much reduced. In contrast, salmine, a basic protein also known to inhibit the actomyosin ATPase, shows less inhibition in the presence of tropomyosin than it does in its absence. Gel electrophoresis data showed that the enhancement of the analog's inhibition by tropomyosin may be related to the analog's promotion of tropomyosin binding to F-actin similar to that reported for Tn-I and that the reduction of salmine inhibition by tropomyosin may be due to the binding of salmine by tropomyosin. At ionic strengths where binding and inhibition of tropomyosin is significant, the analog enhanced inhibition in a manner similar to that reported for whole Tn-I.     

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.     

Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes

Tropomyosin binds end to end along the actin filament. Tropomyosin ends, and the complex they form, are required for actin binding, cooperative regulation of actin filaments by myosin, and binding to the regulatory protein, troponin T. The aim of the work was to understand the isoform and structural specificity of the end-to-end association of tropomyosin. The ability of N-terminal and C-terminal model peptides with sequences of alternate alpha-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.     

Interactions between nebulin-like motifs and thin filament regulatory proteins

Nebulin (600-900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of approximately 35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.     

Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules.

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 gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) 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 troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.     

A 26K fragment of troponin T from rabbit skeletal muscle

A 26K fragment of troponin T, which was produced by endogenous proteases in rabbit skeletal muscle, was isolated by SE-Sephadex column chromatography. This fragment sensitized both superprecipitation and ATPase of actomyosin to calcium ions, to the same extent as troponin T. There was no difference in affinity for tropomyosin between this fragment and troponin T as examined by affinity chromatography. Amino acid analysis showed that this fragment consisted of residues Ala-46-Lys-259 of troponin T. The N-terminal 45 residues of troponin T, therefore, are not essential for the physiological action of troponin T. It was also observed that Ca2+-activated neutral protease digested troponin T into the 26K fragment in the native thin filament, while the protease digested troponin T in a different way in the reconstituted thin filament.