Troponin organization on relaxed and activated thin filaments revealed by electron microscopy and three-dimensional reconstruction

The steric model of muscle regulation holds that at low Ca(2+) concentration, tropomyosin strands, running along thin filaments, are constrained by troponin in an inhibitory position that blocks myosin-binding sites on actin. Ca(2+) activation, releasing this constraint, allows tropomyosin movement, initiating actin-myosin interaction and contraction. Although the different positions of tropomyosin on the thin filament are well documented, corresponding information on troponin has been lacking and it has therefore not been possible to test the model structurally. Here, we show that troponin can be detected on thin filaments and demonstrate how its changing association with actin can control tropomyosin position in response to Ca(2+). To accomplish this, thin filaments were reconstituted with an engineered short tropomyosin, creating a favorable troponin stoichiometry and symmetry for three-dimensional analysis. We demonstrate that in the absence of Ca(2+), troponin bound to both tropomyosin and actin can act as a latch to constrain tropomyosin in a position on actin that inhibits actomyosin ATPase. In addition, we find that on Ca(2+) activation the actin-troponin connection is broken, allowing tropomyosin to assume a second position, initiating actomyosin ATPase and thus permitting contraction to proceed.     

A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle

The regulation of striated muscle contraction involves changes in the interactions of troponin and tropomyosin with actin thin filaments. In resting muscle, myosin-binding sites on actin are thought to be blocked by the coiled-coil protein tropomyosin. During muscle activation, Ca2+ binding to troponin alters the tropomyosin position on actin, resulting in cyclic actin-myosin interactions that accompany muscle contraction. Evidence for this steric regulation by troponin-tropomyosin comes from X-ray data [Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actin-containing filaments of verterbrate striated muscle. Cold Spring Habor Symp. Quant. Biol. 37, 341-352; Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Habor Symp. Quant. Biol. 37, 361-376; Parry, D.A., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33-55] and electron microscope (EM) data [Spudich, J.A., Huxley, H.E., Finch, J., 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632; O'Brien, E.J., Gillis, J.M., Couch, J., 1975. Symmetry and molecular arrangement in paracrystals of reconstituted muscle thin filaments. J. Mol. Biol. 99, 461-475; Lehman, W., Craig, R., Vibert, P., 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65-67] each with its own particular strengths and limitations. Here we bring together some of the latest information from EM analysis of single thin filaments from Pirani et al. [Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L.S., Lehman, W. (2005). Single particle analysis of relaxed and activated muscle thin filaments. J. Mol. Biol. 346, 761-772], with synchrotron X-ray data from non-overlapped muscle fibres to refine the models of the striated muscle thin filament. This was done by incorporating current atomic-resolution structures of actin, tropomyosin, troponin and myosin subfragment-1. Fitting these atomic coordinates to EM reconstructions, we present atomic models of the thin filament that are entirely consistent with a steric regulatory mechanism. Furthermore, fitting the atomic models against diffraction data from skinned muscle fibres, stretched to non-overlap to preclude crossbridge binding, produced very similar results, including a large Ca2+-induced shift in tropomyosin azimuthal location but little change in the actin structure or apparent alteration in troponin position.     

Ca(2+)- and s1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin

The deletion mutant (D234Tm) of rabbit skeletal muscle alpha-tropomyosin, in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing, inhibits the thin filament activated myosin-ATPase activity whether Ca(2+) ion is present or not [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056]. Fluorescence resonance energy transfer (FRET) showed substantial changes in distances between Cys-60 or 250 of troponin T (TnT) and Gln-41 or Cys-374 of actin on wild-type thin filaments corresponding to three states of thin filaments [Kimura et al. (2002) J. Biochem. 132, 93-102]. Troponin T movement on mutant thin filaments reconstituted with D234Tm was compared with that on wild-type thin filaments to understand from which the functional deficiency of mutant thin filaments derives. The Ca(2+)-induced changes in distances between Cys-250 of TnT and Gln-41 or Cys-374 of F-actin were smaller on mutant thin filaments than on wild-type thin filaments. On the other hand, the distances between Cys-60 of TnT and Gln-41 or Cys-374 of F-actin on mutant thin filaments did not change at all regardless of whether Ca(2+) was present. Thus, FRET showed that the Ca(2+)-induced movement of TnT was severely impaired on mutant thin filaments. The rigor binding of myosin subfragment 1 (S1) increased the distances when the thin filaments were fully decorated with S1 in the presence and absence of Ca(2+). However, plots of the extent of S1-incuced movement of TnT against molar ratio of S1 to actin in the presence and absence of Ca(2+) showed that the S1-induced movement of TnT was also impaired on mutant thin filaments. The deficiency of TnT movement on mutant thin filaments causes the altered S1-induced movement of TnI, and mutant thin filaments consequently fail to activate the myosin-ATPase activity even in the presence of Ca(2+).     

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.     

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.     

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.     

Ca(2+)- and S1-induced movement of troponin T on reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca(2+)-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH(2)-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca(2+) binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca(2+), and increased by 2.6 and 5.8 A, respectively upon binding of Ca(2+) to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca(2+)- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca(2+), the rigor S1 binding also increased the distances to the same extent as the presence of Ca(2+) when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca(2+). However, plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca(2+) but sigmoidal curve in the absence of Ca(2+). FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca(2+)-induced or closed, and S1-induced or open states).     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).     

Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament

Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.     

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.     

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.     

Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster

The indirect flight muscles (IFM) of Drosophila melanogaster provide a good genetic system with which to investigate muscle function. Flight muscle contraction is regulated by both stretch and Ca(2+)-induced thin filament (actin + tropomyosin + troponin complex) activation. Some mutants in troponin-I (TnI) and troponin-T (TnT) genes cause a "hypercontraction" muscle phenotype, suggesting that this condition arises from defects in Ca(2+) regulation and actomyosin-generated tension. We have tested the hypothesis that missense mutations of the myosin heavy chain gene, Mhc, which suppress the hypercontraction of the TnI mutant held-up(2) (hdp(2)), do so by reducing actomyosin force production. Here we show that a "headless" Mhc transgenic fly construct that reduces the myosin head concentration in the muscle thick filaments acts as a dose-dependent suppressor of hypercontracting alleles of TnI, TnT, Mhc, and flightin genes. The data suggest that most, if not all, mutants causing hypercontraction require actomyosin-produced forces to do so. Whether all Mhc suppressors act simply by reducing the force production of the thick filament is discussed with respect to current models of myosin function and thin filament activation by the binding of calcium to the troponin complex.     

Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin.

Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation.     

Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C

Understanding the effects of thin and thick filament proteins on the kinetics of Ca(2+) exchange with cardiac troponin C is essential to elucidating the Ca(2+)-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac troponin C at a novel engineered Cys-53 with 2-(4'-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of troponin C upon incorporation into the troponin complex. Neither tropomyosin nor actin alone affected the Ca(2+) binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca(2+) sensitivity ( approximately 7.4-fold) and accelerated the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca(2+) sensitivity ( approximately 6.2-fold) and decreased the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac troponin I with slow skeletal troponin I led to higher Ca(2+) sensitivities and slower Ca(2+) dissociation rates from troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac troponin C to sense and respond to Ca(2+). These results imply that both cross-bridge kinetics and Ca(2+) dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.     

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.     

Effect of caldesmon on the position and myosin-induced movement of smooth muscle tropomyosin bound to actin

It is known that the actin-binding protein caldesmon inhibits actomyosin ATPase activity and might in this way take part in the thin filament regulation of smooth muscle contraction. Although the molecular mechanism of this inhibition is unknown, it is clear that the presence of actin-bound tropomyosin is necessary for full inhibition. Recent evidence also suggests that the myosin-induced movement of tropomyosin plays a key role in regulation. In this work, fluorescence studies provide evidence to show that caldesmon interacts with and alters the position of tropomyosin in a reconstituted actin thin filament and thereby limits the ability of myosin heads to move tropomyosin. Caldesmon interacts with the Cys-190 region in the COOH-terminal half of tropomyosin, resulting in the movement of this part of tropomyosin to a new position on actin. Additionally, this constrains the myosin-induced movement of this region of tropomyosin. On the other hand, caldesmon does not appear to interact with the Cys-36 region in the NH2-terminal half of tropomyosin and neither alters the position of nor significantly constrains the myosin-induced movement of this part of tropomyosin. The ability of caldesmon to limit the myosin-induced movement of tropomyosin provides a possible molecular basis for the inhibitory function of caldesmon. The different movements of the two halves of tropomyosin indicate that actin-bound tropomyosin moves as a flexible molecule and not as a rigid rod. Interestingly, caldesmon, which inhibits tropomyosin's potentiation of actomyosin ATPase activity, moves tropomyosin in one direction, whereas myosin heads, which enhance potentiation, move tropomyosin in the opposite direction.     

Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase.

The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.     

Effects of phosphorylation and mutation R145G on human cardiac troponin I function.

We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.     

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.     

Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments

Tropomyosin movements on thin filaments are thought to sterically regulate muscle contraction, but have not been visualized during active filament sliding. In addition, although 3-D visualization of myosin crossbridges has been possible in rigor, it has been difficult for thick filaments actively interacting with thin filaments. In the current study, using three-dimensional reconstruction of electron micrographs of interacting filaments, we have been able to resolve not only tropomyosin, but also the docking sites for weak and strongly bound crossbridges on thin filaments. In relaxing conditions, tropomyosin was observed on the outer domain of actin, and thin filament interactions with thick filaments were rare. In contracting conditions, tropomyosin had moved to the inner domain of actin, and extra density, reflecting weakly bound, cycling myosin heads, was also detected, on the extreme periphery of actin. In rigor conditions, tropomyosin had moved further on to the inner domain of actin, and strongly bound myosin heads were now observed over the junction of the inner and outer domains. We conclude (1) that tropomyosin movements consistent with the steric model of muscle contraction occur in interacting thick and thin filaments, (2) that myosin-induced movement of tropomyosin in activated filaments requires strongly bound crossbridges, and (3) that crossbridges are bound to the periphery of actin, at a site distinct from the strong myosin binding site, at an early stage of the crossbridge cycle.