Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin.

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding.

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Comparative studies on troponin, a Ca²âº-dependent regulator of muscle contraction, in striated and smooth muscles of protochordates.

Troponin is well known as a Ca(2+)-dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin-myosin interaction at low Ca(2+) concentrations, and Ca(2+) at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca(2+)-dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca(2+). Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals.

Structural basis for the regulation of muscle contraction by troponin and tropomyosin.

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca2+ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca2+ binding to TnC (the Ca2+ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the "inhibitory" subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca2+ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction.

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by approximately 0.1% upon activation relative to the relaxing state and increased by approximately 0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca(2+)-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca(2+)-binding and the second induced by actomyosin interaction.

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.

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.

Crystal structure of troponin and the molecular mechanism of muscle regulation.

Troponin plays a central role in the regulation of skeletal and cardiac muscle contraction. The protein consists of three polypeptide chains (TnT, TnI and TnC) and is located on polymerized actin together with tropomyosin, forming muscle thin filament. We have determined the molecular structures of the core domains (relative molecular mass of 46,000 and 52,000) of human cardiac troponin in the Ca2+-saturated form by X-ray crystallography. Analysis of the four structures derived from the two crystal forms reveals that the core domain is further divided into sub-domains, connected by linkers, making the entire molecule highly flexible. The structures of the troponin ternary complex suggests that the Ca2+-binding to the regulatory TnC site displaces the carboxyl-terminal portion of TnI from actin/tropomyosin, thereby altering mobility and/or flexibility of the troponin/tropomyosin strand on the actin filament. These Ca2+-dependent changes in the properties of the tropomyosin strand on the actin filament may in turn alter accessibility of myosin heads (motor protein) to the actin filament.

Single particle analysis of relaxed and activated muscle thin filaments.

The movement of tropomyosin from actin's outer to its inner domain plays a key role in sterically regulating muscle contraction. This movement, from a low Ca2+ to a Ca2+-induced position has been directly demonstrated by electron microscopy and helical reconstruction. Solution studies, however, suggest that tropomyosin oscillates dynamically between these positions at all Ca2+ levels, and that it is the position of this equilibrium that is controlled by Ca2+. Helical reconstruction reveals only the average position of tropomyosin on the filament, and not information on the local dynamics of tropomyosin in any one Ca2+ state. We have therefore used single particle analysis to analyze short filament segments to reveal local variations in tropomyosin behavior. Segments of Ca2+-free and Ca2+ treated thin filaments were sorted by cross-correlation to low and high Ca2+ models of the thin filament. Most segments from each data set produced reconstructions matching those previously obtained by helical reconstruction, showing low and high Ca2+ tropomyosin positions for low and high Ca2+ filaments. However, approximately 20% of segments from Ca2+-free filaments fitted best to the high Ca2+ model, yielding a corresponding high Ca2+ reconstruction. Conversely, approximately 20% of segments from Ca2+-treated filaments fitted best to the low Ca2+ model and produced a low Ca2+ reconstruction. Hence, tropomyosin position on actin is not fixed in either Ca2+ state. These findings provide direct structural evidence for the equilibration of tropomyosin position in both high and low Ca2+ states, and for the concept that Ca2+ controls the position of this equilibrium. This flexibility in the localization of tropomyosin may provide a means of sterically regulating contraction at low energy cost.

Ca(2+)-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy.

Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.

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.

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.

Isolation, electron microscopic imaging, and 3-D visualization of native cardiac thin myofilaments.

An increasing number of cardiac diseases are currently pinpointed to reside at the level of the thin myofilaments (e.g., cardiomyopathies, reperfusion injury). Hence the aim of our study was to develop a new method for the isolation of mammalian thin myofilaments suitable for subsequent high-resolution electron microscopic imaging. Native cardiac thin myofilaments were extracted from glycerinated porcine myocardial tissue in the presence of protease inhibitors. Separation of thick and thin myofilaments was achieved by addition of ATP and several centrifugation steps. Negative staining and subsequent conventional and scanning transmission electron microscopy (STEM) of thin myofilaments permitted visualization of molecular details; unlike conventional preparations of thin myofilaments, our method reveals the F-actin moiety and allows direct recognition of thin myofilament-associated porcine cardiac troponin complexes. They appear as "bulges" at regular intervals of approximately 36 nm along the actin filaments. Protein analysis using SDS-polyacrylamide gel electrophoresis revealed that only approximately 20% troponin I was lost during the isolation procedure. In a further step, 3-D helical reconstructions were calculated using STEM dark-field images. These 3-D reconstructions will allow further characterization of molecular details, and they will be useful for directly visualizing molecular alterations related to diseased cardiac thin myofilaments (e.g., reperfusion injury, alterations of Ca2+-mediated tropomyosin switch).

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.

Structure of the insect troponin complex.

Isolated troponin-tropomyosin complex from Lethocerus indicus asynchronous flight muscle forms paracrystals on a positively charged lipid monolayer. Single particle analysis was carried out on individual complexes selected from electron micrographs of negatively stained paracrystals. By a combination of correlation and classification techniques, different average projections of the object were obtained. An initial three-dimensional model was calculated by determining the Euler angles for the different views using a common line approach. This starting model was then used as a reference for the further three-dimensional refinement of the model using the original data set. The refined model of the troponin complex has a diameter of approximately 90 A and a volume corresponding with a molecular mass of about 120 kDa for the globular domain. The resolution of the reconstruction was determined to be 32 A using the differential phase residual method and 26 A using the Fourier shell correlation criterion.

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 of the Lethocerus troponin-tropomyosin complex as determined by electron microscopy.

Native troponin-tropomyosin complex was isolated from Lethocerus indicus indirect flight muscle and tested for function. It was shown by rotary shadowing and by forming paracrystals on monolayers that the regulatory complex consists of a troponin head region approximately 130 A in diameter and a 400-A-long troponin T-tropomyosin tail. The complex forms paracrystals at the air-water interface on a positively charged monolayer. The globular head packs in rows 380 A apart which are bridged by the tail domain. Filamentous paracrystals were obtained by adding Mg2+ ions to the troponin-tropomyosin sample. These showed globular domains arranged in a regular pattern along "ribbon"-like filaments. The spacing of the repeat was determined to be 380 A.

Immunocytochemical electron microscopic study and Western blot analysis of troponin in striated muscle of the fruit fly Drosophila melanogaster and in several muscle cell types of the earthworm Eisenia foetida.

BACKGROUND: There is little information about troponin in invertebrate muscles, and no previous references to this protein in annelid muscles have been found. The aim of this paper was to study the presence and distribution of troponin in different muscle cell types from the earthworm Eisenia foetida (the muscular body wall, and the inner and outer muscular layer of the pseudoheart). These results were compared with those obtained in the transversely striated muscle of Drosophila melanogaster and in skeletal and smooth muscles of the mouse. METHODS: Immunocytochemical electron microscopic study and Western blot analysis using anti-TnT antibodies were employed in this study. RESULTS: Troponin immunoreaction was detected in the mouse skeletal muscle, the fly flight muscle, and earthworm obliquely striated muscles (body wall musculature and inner muscular layer of the pseudoheart). Immunolabeling for TnT in all these muscle cells appeared in moderate amounts at any point along the sarcomere length, except for the central zone of the A band (H band). This suggests that troponin molecules were located along the thin filaments. The density of immunogold particles was similar in the three muscles, and thus the amount of troponin in each muscle type was proportional to the number and length of actin filaments in each. Troponin was found in neither the mouse smooth muscle nor the outer muscular layer of the earthworm pseudoheart. The latter muscle showed an ultrastructural pattern that was intermediate between obliquely striated and smooth muscle. The estimated molecular weight for TnT in the earthworm was 55 kDa; this is higher than the weight of this protein in the mouse skeletal muscle (40 kDa) but similar to that of the D. melanogaster muscle (52 kDa). CONCLUSIONS: Troponin is present in both types of striated muscle (transversely striated and obliquely striated) of the earthworm with a distribution that is very similar to that observed in the mammalian striated muscle. As in vertebrates, troponin is absent in the smooth muscle of the earthworm. Discrepancies in the classification of some invertebrate muscles are common in the literature, and the use of distinctive markers, such as troponin, may improve our understanding of the nature and properties of many invertebrate muscles showing an ultrastructural pattern that does not resemble any of the classic muscle types.