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.     

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.     

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.     

Cardiomyopathic tropomyosin mutations that increase thin filament Ca2+ sensitivity and tropomyosin N-domain flexibility

The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of alpha-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10 degrees C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 degrees C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0-1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca2+ binding sites of thin filament in two settings: Ca2+-dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca2+ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca2+ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca2+ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca2+ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.     

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.     

The effects of troponin T fragments T1 and T2 on the binding of nonpolymerizable tropomyosin to F-actin in the presence and absence of troponin I and troponin C

Using a nonpolymerizable form of tropomyosin (NPTM) we have investigated the interactions between the T1 (residues 1-158) and T2 (residues 159-259) regions of troponin T and the other components of the thin filament at 50 mM KCl +/- Ca2+. Under these conditions the binding of NPTM to F-actin is fully restored by whole troponin (+/- Ca2+), and in each case, retains a residual degree of cooperativity as demonstrated by Scatchard and Hill plots. Fragment T2 alone had a small inductive effect on the interaction of NPTM with F-actin. In the presence of troponin I, this interaction is increased to a level which exceeds that observed with either component alone. The effects of T2 and troponin I are moderately (-Ca2+) and markedly (+Ca2+) reduced by troponin C. While fragment T1 alone did not promote induction, it accentuated the effects of T2 and troponin I. Since T1 does not interact with T2 or troponin I but does interact weakly with the NH2 terminus of tropomyosin and can be expected to bind weakly at the residual interaction site(s) at the COOH terminus of NPTM, the observed effects of T1 have been ascribed to the linking of neighboring NPTM molecules at their ends.     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

Cooperative interactions between troponin molecules bound to the cardiac thin filament

Striated muscle thin filaments contain many troponin molecules, which contact each other indirectly via tropomyosin and actin. Such allosteric interactions between troponin molecules may be responsible for cooperative Ca2+ binding to the regulatory sites of the cardiac thin filament (Tobacman, L. S., and Sawyer, D. S. (1990) J. Biol. Chem. 265, 931-939). To test whether thin filament-bound troponin molecules interact, we studied the competitive binding of troponin and troponin T-troponin I (an inhibitory complex lacking the Ca2+ binding subunit troponin C) to actin-tropomyosin. The relative affinities of these two forms of troponin for the thin filament depended upon their relative concentrations. Under conditions where total binding was saturated, each form binds with greater apparent affinity to sites that have similar neighbors. A theoretical model for competitive binding of two ligands to interacting sites on a linear lattice was developed and fit to the data. Surprisingly, energetically unfavorable interactions occurred between adjacent troponin and troponin T-troponin I molecules not only in the presence of Ca2+, but also in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and/or myosin subfragment 1. Removal of Ca2+ strengthened the affinity of troponin for the thin filament less than 50%. These results suggest that, even in the absence of myosin, long range allosteric interactions occur between troponin molecules. The detailed involvement of tropomyosin and actin in these interactions remains to be established.     

An atomic model of the thin filament in the relaxed and Ca2+-activated states

Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the "core domain" of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.     

Functional effects of congenital myopathy-related mutations in gamma-tropomyosin gene

Missense mutations in human TPM3 gene encoding γ-tropomyosin expressed in slow muscle type 1 fibers, were associated with three types of congenital myopathies-nemaline myopathy, cap disease and congenital fiber type disproportion. Functional effects of the following substitutions: Leu100Met, Ala156Thr, Arg168His, Arg168Cys, Arg168Gly, Lys169Glu, and Arg245Gly, were examined in biochemical assays using recombinant tropomyosin mutants and native proteins isolated from skeletal muscle. Most, but not all, mutations decreased the affinity of tropomyosin for actin alone and in complex with troponin (±Ca(2+)). All studied tropomyosin mutants reduced Ca-induced activation but had no effect on the inhibition of actomyosin cross-bridges. Ca(2+)-sensitivity of the actomyosin interactions, as well as cooperativity of myosin-induced activation of the thin filament was affected by individual tropomyosin mutants with various degrees. Decreased motility of the reconstructed thin filaments was a result of combined functional defects caused by myopathy-related tropomyosin mutants. We conclude that muscle weakness and structural abnormalities observed in TPM3-related congenital myopathies result from reduced capability of the thin filament to fully activate actin-myosin cross-bridges.     

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.     

A new model of cooperative myosin-thin filament binding

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.     

M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end

Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin–tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin''s reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T''s N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin–tropomyosin binding and weaker tropomyosin–actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin–thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM.     

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Independent functions for the N- and C-termini in the overlap region of tropomyosin

Tropomyosin (TM) is a coiled-coil that binds head-to-tail along the helical actin filament. The ends of 284-residue tropomyosins are believed to overlap by about nine amino acids. The present study investigates the function of the N- and C-terminal overlap regions. Recombinant tropomyosins were produced in Escherichia coli in which nine amino acids were truncated from the N-terminal, C-terminal, or both ends of striated muscle alpha-tropomyosin (TM9a) and TM2 (TM9d), a nonmuscle alpha-tropomyosin expressed in many cells. The two isoforms are identical except for the C-terminal 27 amino acids encoded by exon 9a (striated) or exon 9d (TM2). Removal of either end greatly reduces the actin affinity of both tropomyosins in all conditions and the cooperativity with which myosin promotes tropomyosin binding to actin in the open state. N-Terminal truncations generally are more deleterious than C-terminal truncations. With TM9d, truncation of the N-terminus is as deleterious as both for myosin S1-induced binding. None of the TM9d variants binds well to actin with troponin (+/-Ca(2+)). TM9a with the truncated N-terminus binds more weakly to actin with troponin (-Ca(2+)) than when the C-terminus is removed but more strongly than when both ends are removed; the actin binding of all three forms is cooperative. The results show that the ends of TM9a, though important, are not required for cooperative function and suggest they have independent functions beyond formation of an overlap complex. The nonadditivity of the TM9d truncations suggests that the ends may primarily function as a complex in this isoform. A surprising result is that all variants bound with the same affinity, and noncooperatively, to actin saturated with myosin S1. Evidently, end-to-end interactions are not required for high-affinity binding to acto-myosin S1.     

The amino terminus of muscle tropomyosin is a major determinant for function

The amino-terminal region of muscle tropomyosin is highly conserved among muscle and 284-residue non-muscle tropomyosins. Analysis of fusion and nonfusion striated alpha-tropomyosins and a mutant in which residues 1-9 have been deleted has shown that the amino terminus is crucial for function. The presence of 80 amino acids of a nonstructural influenza virus protein (NS1) on the amino terminus of tropomyosin allows magnesium-independent binding of tropomyosin to actin. The fusion tropomyosin inhibits the actomyosin S1 ATPase at all myosin S1 concentrations tested, indicating that the presence of the fusion peptide prevents myosin S1 from switching the actin filament from the inhibited to the potentiated state. Nonfusion tropomyosin, an unacetylated form, has no effect on the actomyosin S1 ATPase, though it regulates normally with troponin. Deletion of residues 1-9, which are believed to overlap with the carboxyl-terminal end of tropomyosin in the thin filament, results in loss of tropomyosin function. The mutant is unable to bind to actin, in the presence and absence of troponin, and it has no regulatory function. The removal of the first 9 residues of tropomyosin is much more deleterious than removal of the last 11 by carboxypeptidase digestion. We suggest that the structure of the amino-terminal region and acetylation of the initial methionine are crucial for tropomyosin function.     

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.     

Folding and function of the troponin tail domain. Effects of cardiomyopathic troponin T mutations

Troponin contains a globular Ca(2+)-binding domain and an elongated tail domain composed of the N terminus of subunit troponin T (TnT). The tail domain anchors troponin to tropomyosin and actin, modulates myosin function, and is a site of cardiomyopathy-inducing mutations. Critical interactions between tropomyosin and troponin are proposed to depend on tail domain residues 112-136, which are highly conserved across phyla. Most cardiomyopathy mutations in TnT flank this region. Three such mutations were examined and had contrasting effects on peptide TnT-(1-156), promoting folding and thermal stability assessed by circular dichroism (F110I) or weakening folding and stability (T104V and to a small extent R92Q). Folding of both TnT-(1-156) and whole troponin was promoted by replacing bovine TnT Thr-104 with human TnT Ala-104, further indicating the importance of this cardiomyopathy site residue for protein folding. Mutation F110I markedly stabilized the troponin tail but weakened binding of holo-troponin to actin-tropomyosin 8-fold, suggesting that loss of flexibility impairs troponin tail function. The effect of the F110I mutation on troponin-tropomyosin binding to actin was much less, indicating this flexibility is particularly important for the interactions of troponin with tropomyosin. We suggest that most cardiomyopathic mutations in the troponin tail alter muscle function indirectly, by perturbing interactions between troponin and tropomyosin requisite for the complex effects of these proteins on myosin.