Myopathy-causing mutation R91P in the TPM3 gene drastically impairs structural and functional properties of slow skeletal muscle tropomyosin γß-heterodimer.

Tropomyosin (Tpm) is a regulatory actin-binding protein involved in Ca2+ activation of contraction of striated muscle. In human slow skeletal muscles, two distinct Tpm isoforms, γ and ß, are present. They interact to form three types of dimeric Tpm molecules: γγ-homodimers, γß-heterodimers, or ßß-homodimers, and a majority of the molecules are present as γß-Tpm heterodimers. Point mutation R91P within the TPM3 gene encoding γ-Tpm is linked to the condition known as congenital fiber-type disproportion (CFTD), which is characterized by severe muscle weakness. Here, we investigated the influence of the R91P mutation in the γ-chain on the properties of the γß-Tpm heterodimer. We found that the R91P mutation impairs the functional properties of γß-Tpm heterodimer more severely than those of earlier studied γγ-Tpm homodimer carrying this mutation in both γ-chains. Since a significant part of Tpm molecules in slow skeletal muscle is present as γß-heterodimers, our results explain why this mutation leads to muscle weakness in CFTD.

Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation.

We characterized a novel genetic variant c.292G > A (p.E98K) in the TPM1 gene encoding cardiac tropomyosin 1.1 isoform (Tpm1.1), found in a proband with a phenotype of complex cardiomyopathy with conduction dysfunction and slow progressive neuromuscular involvement. To understand the molecular mechanism by which this mutation impairs cardiac function, we produced recombinant Tpm1.1 carrying an E98K substitution and studied how this substitution affects the structure of the Tpm1.1 molecule and its functional properties. The results showed that the E98K substitution in the N-terminal part of the Tpm molecule significantly destabilizes the C-terminal part of Tpm, thus indicating a long-distance destabilizing effect of the substitution on the Tpm coiled-coil structure. The E98K substitution did not noticeably affect Tpm's affinity for F-actin but significantly impaired Tpm's regulatory properties. It increased the Ca2+ sensitivity of the sliding velocity of regulated thin filaments over cardiac myosin in an in vitro motility assay and caused an incomplete block of the thin filament sliding at low Ca2+ concentrations. The incomplete motility block in the absence of Ca2+ can be explained by the loosening of the Tpm interaction with troponin I (TnI), thus increasing Tpm mobility on the surface of an actin filament that partially unlocks the myosin binding sites. This hypothesis is supported by the molecular dynamics (MD) simulation that showed that the E98 Tpm residue is involved in hydrogen bonding with the C-terminal part of TnI. Thus, the results allowed us to explain the mechanism by which the E98K Tpm mutation impairs sarcomeric function and myocardial relaxation.

Structural and Functional Properties of Kappa Tropomyosin.

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

Molecular Dynamics Assessment of Mechanical Properties of the Thin Filaments in Cardiac Muscle.

Contraction of cardiac muscle is regulated by Ca2+ ions via regulatory proteins, troponin (Tn), and tropomyosin (Tpm) associated with the thin (actin) filaments in myocardial sarcomeres. The binding of Ca2+ to a Tn subunit causes mechanical and structural changes in the multiprotein regulatory complex. Recent cryo-electron microscopy (cryo-EM) models of the complex allow one to study the dynamic and mechanical properties of the complex using molecular dynamics (MD). Here we describe two refined models of the thin filament in the calcium-free state that include protein fragments unresolved by cryo-EM and reconstructed using structure prediction software. The parameters of the actin helix and the bending, longitudinal, and torsional stiffness of the filaments estimated from the MD simulations performed with these models were close to those found experimentally. However, problems revealed from the MD simulation suggest that the models require further refinement by improving the protein-protein interaction in some regions of the complex. The use of relatively long refined models of the regulatory complex of the thin filament allows one to perform MD simulation of the molecular mechanism of Ca2+ regulation of contraction without additional constraints and study the effects of cardiomyopathy-associated mutation of the thin filament proteins of cardiac muscle.

Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle.

Electron microscopy (EM) shows that myosin heads in thick filaments isolated from striated muscles interact with each other and with the myosin tail under relaxing conditions. This "interacting-heads motif" (IHM) is highly conserved across the animal kingdom and is thought to be the basis of the super-relaxed state. However, a recent X-ray modeling study concludes, contrary to expectation, that the IHM is not present in relaxed intact muscle. We propose that this conclusion results from modeling with a thick filament 3D reconstruction in which the myosin heads have radially collapsed onto the thick filament backbone, not from absence of the IHM. Such radial collapse, by about 3-4 nm, is well established in EM studies of negatively stained myosin filaments, on which the reconstruction was based. We have tested this idea by carrying out similar X-ray modeling and determining the effect of the radial position of the heads on the goodness of fit to the X-ray pattern. We find that, when the IHM is modeled into a thick filament at a radius 3-4 nm greater than that modeled in the recent study, there is good agreement with the X-ray pattern. When the original (collapsed) radial position is used, the fit is poor, in agreement with that study. We show that modeling of the low-angle region of the X-ray pattern is relatively insensitive to the conformation of the myosin heads but very sensitive to their radial distance from the filament axis. We conclude that the IHM is sufficient to explain the X-ray diffraction pattern of intact muscle when placed at the appropriate radius.

Tropomyosin pseudo-phosphorylation can rescue the effects of cardiomyopathy-associated mutations.

We applied various methods to investigate how mutations S283D and S61D that mimic phosphorylation of tropomyosin (Tpm) affect structural and functional properties of cardiac Tpm carrying cardiomyopathy-associated mutations in different parts of its molecule. Using differential scanning calorimetry and molecular dynamics, we have shown that the S61D mutation (but not the S283 mutation) causes significant destabilization of the N-terminal part of the Tpm molecule independently of the absence or presence of cardiomyopathy-associated mutations. Our results obtained by cosedimentation of Tpm with F-actin demonstrated that both S283D and S61D mutations can reduce or even eliminate undesirable changes in Tpm affinity for F-actin caused by some cardiomyopathy-associated mutations. The results indicate that Tpm pseudo-phosphorylation by mutations S283D or S61D can rescue the effects of mutations in the TPM1 gene encoding a cardiac isoform of Tpm that lead to the development of such severe inherited heart diseases as hypertrophic or dilated cardiomyopathies.

Impact of A134 and E218 Amino Acid Residues of Tropomyosin on Its Flexibility and Function.

Tropomyosin (Tpm) is one of the major actin-binding proteins that play a crucial role in the regulation of muscle contraction. The flexibility of the Tpm molecule is believed to be vital for its functioning, although its role and significance are under discussion. We choose two sites of the Tpm molecule that presumably have high flexibility and stabilized them with the A134L or E218L substitutions. Applying differential scanning calorimetry (DSC), molecular dynamics (MD), co-sedimentation, trypsin digestion, and in vitro motility assay, we characterized the properties of Tpm molecules with these substitutions. The A134L mutation prevented proteolysis of Tpm molecule by trypsin, and both substitutions increased the thermal stability of Tpm and its bending stiffness estimated from MD simulation. None of these mutations affected the primary binding of Tpm to F-actin; still, both of them increased the thermal stability of the actin-Tpm complex and maximal sliding velocity of regulated thin filaments in vitro at a saturating Ca2+ concentration. However, the mutations differently affected the Ca2+ sensitivity of the sliding velocity and pulling force produced by myosin heads. The data suggest that both regions of instability are essential for correct regulation and fine-tuning of Ca2+-dependent interaction of myosin heads with F-actin.

Functional outcomes of structural peculiarities of striated muscle tropomyosin.

Tropomyosin is a dimer coiled-coil actin-binding protein. Adjacent tropomyosin molecules connect each other 'head-to-tail' via an overlap junction and form a continuous strand that winds around an actin filament and controls the actin-myosin interaction. High cooperativity of muscle contraction largely depends on tropomyosin characteristics. Here we summarise experimental evidence that local peculiarities of tropomyosin structure have long-range effects and determine functional properties of the strand, including changes in its bending stiffness and interaction with actin and myosin. Point mutations and posttranslational modifications help to probe the roles of the conserved 'non-canonical' residues, clusters of stabilising and destabilising core residues, and core gap in tropomyosin function. The data suggest that tropomyosin structural lability including a diversity of homo- and heterodimers of different isoforms provide a balance of stiffness, flexibility, and strength of interaction with partner sarcomere proteins necessary for fine-tuning of Ca2+ regulation in various types of striated muscles.

Cardiomyopathy-associated mutations in tropomyosin differently affect actin-myosin interaction at single-molecule and ensemble levels.

In the heart, mutations in the TPM1 gene encoding the α-isoform of tropomyosin lead, in particular, to the development of hypertrophic and dilated cardiomyopathies. We compared the effects of hypertrophic, D175N and E180G, and dilated, E40K and E54K, cardiomyopathy mutations in TPM1 gene on the properties of single actin-myosin interactions and the characteristics of the calcium regulation in an ensemble of myosin molecules immobilised on a glass surface and interacting with regulated thin filaments. Previously, we showed that at saturating Ca2+ concentration the presence of Tpm on the actin filament increases the duration of the interaction. Here, we found that the studied Tpm mutations differently affected the duration: the D175N mutation reduced it compared to WT Tpm, while the E180G mutation increased it. Both dilated mutations made the duration of the interaction even shorter than with F-actin. The duration of the attached state of myosin to the thin filament in the optical trap did not correlate to the sliding velocity of thin filaments and its calcium sensitivity in the in vitro motility assay. We suppose that at the level of the molecular ensemble, the cooperative mechanisms prevail in the manifestation of the effects of cardiomyopathy-associated mutations in Tpm.

The effects of cardiomyopathy-associated mutations in the head-to-tail overlap junction of α-tropomyosin on its properties and interaction with actin.

Tropomyosin (Tpm) plays a crucial role in the regulation of muscle contraction by controlling actin-myosin interaction. Tpm coiled-coil molecules bind each other via overlap junctions of their N- and C-termini and form a semi-rigid strand that binds the helical surface of an actin filament. The high bending stiffness of the strand is essential for high cooperativity of muscle regulation. Point mutations M8R and K15N in the N-terminal part of the junction and the A277V one in the C-terminal part are associated with dilated cardiomyopathy, while the M281T and I284V mutations are related to hypertrophic cardiomyopathy. To reveal molecular mechanism(s) underlying these pathologies, we studied the properties of recombinant Tpm carrying these mutations using several experimental approaches and molecular dynamic simulation of the junction. The M8R and K15N mutations weakened the interaction between the N- and C-termini of Tpm in the overlap junction and reduced the Tpm affinity for actin. These changes possibly led to a reduction in the regulation cooperativity. The C-terminal mutations caused only small and controversial changes in properties of Tpm and its complex with actin. Their involvement in disease phenotype is possibly caused by interaction with other sarcomere proteins.

Effects of an Interchain Disulfide Bond on Tropomyosin Structure: A Molecular Dynamics Study.

Tropomyosin (Tpm) is a coiled-coil actin-binding dimer protein that participates in the regulation of muscle contraction. Both Tpm chains contain Cys190 residues which are normally in the reduced state, but form an interchain disulfide bond in failing heart. Changes in structural and functional properties of Tpm and its complexes with actin upon disulfide cross-linking were studied using various experimental methods. To understand the molecular mechanism underlying these changes and to reveal the possible mechanism of the involvement of the cross-linking in heart failure, molecular dynamics (MD) simulations of the middle part of Tpm were performed in cross-linked and reduced states. The cross-linking increased bending stiffness of Tpm assessed from MD trajectories at 27 °C in agreement with previous experimental observations. However, at 40 °C, the cross-linking caused a decrease in Tpm stiffness and a significant reduction in the number of main chain hydrogen bonds in the vicinity of residues 133 and 134. These data are in line with observations showing enhanced thermal unfolding of the least stable part of Tpm at 30⁻40 °C and accelerated trypsin cleavage at residue 133 at 40 °C (but not at 27 °C) upon cross-linking. These results allow us to speculate about the possible mechanism of involvement of Tpm cross-linking to heart failure pathogenesis.

Functional role of the core gap in the middle part of tropomyosin.

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein playing an essential role in the regulation of muscle contraction. The middle part of the Tpm molecule has some specific features, such as the presence of noncanonical residues as well as a substantial gap at the interhelical interface, which are believed to destabilize a coiled-coil and impart structural flexibility to this part of the molecule. To study how the gap affects structural and functional properties of α-striated Tpm (the Tpm1.1 isoform that is expressed in cardiac and skeletal muscles) we replaced large conserved apolar core residues located at both sides of the gap with smaller ones by mutations M127A/I130A and M141A/Q144A. We found that in contrast with the stabilizing substitutions D137L and G126R studied earlier, these substitutions have no appreciable influence on thermal unfolding and domain structure of the Tpm molecule. They also do not affect actin-binding properties of Tpm. However, they strongly increase sliding velocity of regulated actin filaments in an in vitro motility assay and cause an oversensitivity of the velocity to Ca2+ similar to the stabilizing substitutions D137L and G126R. Molecular dynamics shows that the substitutions studied here increase bending stiffness of the coiled-coil structure of Tpm, like that of G126R/D137L, probably due to closure of the interhelical gap in the area of the substitutions. Our results clearly indicate that the conserved middle part of Tpm is important for the fine tuning of the Ca2+ regulation of actin-myosin interaction in muscle.

The Closed State of the Thin Filament Is Not Occupied in Fully Activated Skeletal Muscle.

Muscle contraction is powered by actin-myosin interaction controlled by Ca2+ via the regulatory proteins troponin (Tn) and tropomyosin (Tpm), which are associated with actin filaments. Tpm forms coiled-coil dimers, which assemble into a helical strand that runs along the whole ∼1 µm length of a thin filament. In the absence of Ca2+, Tn that is tightly bound to Tpm binds actin and holds the Tpm strand in the blocked, or B, state, where Tpm shields actin from the binding of myosin heads. Ca2+ binding to Tn releases the Tpm from actin so that it moves azimuthally around the filament axis to a closed, or C, state, where actin is partially available for weak binding of myosin heads. Upon transition of the weak actin-myosin bond into a strong, stereo-specific complex, the myosin heads push Tpm strand to the open, or O, state allowing myosin binding sites on several neighboring actin monomers to become open for myosin binding. We used low-angle x-ray diffraction at the European Synchrotron Radiation Facility to check whether the O- to C-state transition in fully activated fibers of fast skeletal muscle of the rabbit occurs during transition from isometric contraction to shortening under low load. No decrease in the intensity of the second actin layer line at reciprocal radii in the range of 0.15-0.275 nm-1 was observed during shortening suggesting that an azimuthal Tpm movement from the O- to C-state does not occur, although during shortening muscle stiffness is reduced compared to the isometric state, and the intensities of other actin layer lines demonstrate a ∼2-fold decrease in the fraction of myosin heads strongly bound to actin. The data show that a small fraction of actin-bound myosin heads is sufficient for supporting the O-state and, therefore the C-state is not occupied in fully activated skeletal muscle that produces mechanical work at low load.

Tropomyosin movement is described by a quantitative high-resolution model of X-ray diffraction of contracting muscle.

Contraction of skeletal and cardiac muscle is controlled by Ca2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca2+, Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the three-state model (McKillop and Geeves Biophys J 65:693-701, 1993), upon Ca2+ binding to Tn, Tpm rotates about the filament axis to a 'closed state' where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an 'open' position where neighboring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low-angle X-ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of X-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much-improved fit to the experimental data obtained from rabbit muscle fibers in relaxed and rigor states than previous lower-resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 to 0.3 nm-1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin.

Stabilizing the central part of tropomyosin increases the bending stiffness of the thin filament.

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.

Why muscle is an efficient shock absorber.

Skeletal muscles power body movement by converting free energy of ATP hydrolysis into mechanical work. During the landing phase of running or jumping some activated skeletal muscles are subjected to stretch. Upon stretch they absorb body energy quickly and effectively thus protecting joints and bones from impact damage. This is achieved because during lengthening, skeletal muscle bears higher force and has higher instantaneous stiffness than during isometric contraction, and yet consumes very little ATP. We wish to understand how the actomyosin molecules change their structure and interaction to implement these physiologically useful mechanical and thermodynamical properties. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch compared to those during isometric contraction at physiological temperature using synchrotron radiation. The intensities of the off-meridional layer lines and fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that upon stretch the fraction of actin-bound myosin heads is higher than during isometric contraction. On the other hand, the intensities of the actin layer lines are lower than during isometric contraction. Taken together, these results suggest that during stretch, a significant fraction of actin-bound heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. As the strong or stereo-specific myosin binding to actin is necessary for actin activation of the myosin ATPase, this finding explains the low metabolic cost of energy absorption by muscle during the landing phase of locomotion.

The fraction of myosin motors that participate in isometric contraction of rabbit muscle fibers at near-physiological temperature.

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca(2+) at 4°C were heated to 31-34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41-43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ~6.3 pN.

Myosin heads contribute to the maintenance of filament order in relaxed rabbit muscle.

Raising the temperature of rabbit skeletal muscle from ∼0°C to ∼20°C has been shown to enhance the helical organization of the myosin heads and to change the intensities of the 10 and 11 equatorial reflections. We show here by time-resolved x-ray diffraction combined with temperature jump that the movement of the heads to enhance the organized myosin helix occurs at the same fast rate as the change in the intensities of the equatorial reflections. However, model calculations indicate that the change in the equatorials cannot be explained simply in terms of the movement of myosin heads. Analysis of electron micrographs of transverse sections of relaxed muscle fibers cryofixed at ∼5°C and ∼35°C shows that in addition to the reorganization of the heads the thin and thick filaments are less constrained to their positions in the hexagonal filament lattice in the warm muscle than in the cold. Incorporating the changes in filament order in model calculations reconciles these with the observed changes in equatorial reflections. We suggest the thin filaments in the cold muscle are boxed into their positions by the thermal movement of the disordered myosin heads. In the warmer muscle, the packed-down heads leave the thin filaments more room to diffuse laterally.

Insight into the actin-myosin motor from x-ray diffraction on muscle.

The origin of reflections in the x-ray diffraction pattern from striated muscle and their use for understanding the structural organization of the contractile machinery are presented and discussed. Results of x-ray diffraction experiments obtained by a number of research groups using a variety of protocols revealed structural changes in contracting muscles which are interpreted in terms of molecular movements that underlie force generation. Some of these data are in line with the widely accepted 'lever arm' hypothesis which links force generation to a tilt of the light chain domain of the myosin head with respect to its motor domain. However, changes in the layer line intensities observed in response to various perturbations cannot be explained by tilting of the lever arm. Such changes, first revealed in response to temperature jumps, are interpreted as a transition of non-stereo-specifically attached myosin heads to a stereo-specifically bound state. The new 'roll and lock' model considers force-generation as a two-stage process: initial stereo-specific locking of myosin heads on actin is followed by the lever arm tilt.

Direct modeling of X-ray diffraction pattern from contracting skeletal muscle.

A direct modeling approach was used to quantitatively interpret the two-dimensional x-ray diffraction patterns obtained from contracting mammalian skeletal muscle. The dependence of the calculated layer line intensities on the number of myosin heads bound to the thin filaments, on the conformation of these heads and on their mode of attachment to actin, was studied systematically. Results of modeling are compared to experimental data collected from permeabilized fibers from rabbit skeletal muscle contracting at 5 degrees C and 30 degrees C and developing low and high isometric tension, respectively. The results of the modeling show that: i), the intensity of the first actin layer line is independent of the tilt of the light chain domains of myosin heads and can be used as a measure of the fraction of myosin heads stereospecifically attached to actin; ii), during isometric contraction at near physiological temperature, the fraction of these heads is approximately 40% and the light chain domains of the majority of them are more perpendicular to the filament axis than in rigor; and iii), at low temperature, when isometric tension is low, a majority of the attached myosin heads are bound to actin nonstereospecifically whereas at high temperature and tension they are bound stereospecifically.