Mechanochemical consequences of myopathy-linked mutations in Tpm2.2 on striated muscle contractility.

Tropomyosin (Tpm) is an actin-binding protein central to muscle contraction regulation. The Tpm sequence consists of periodic repeats corresponding to seven actin-binding sites, further divided in two functionally distinct halves. To clarify the importance of the first and second halves of the actin-binding periods in regulating the interaction of myosin with actin, we introduced hypercontractile mutations D20H, E181K located in the N-terminal halves of periods 1 and 5 and hypocontractile mutations E41K, N202K located in the C-terminal halves of periods 1 and 5 of the skeletal muscle Tpm isoform Tpm2.2. Wild-type and mutant Tpms displayed similar actin-binding properties, however, as revealed by FRET experiments, the hypercontractile mutations affected the binding geometry and orientation of Tpm2.2 on actin, causing a stimulation of myosin motor performance. Contrary, the hypocontractile mutations led to an inhibition of both, actin activation of the myosin ATPase and motor activity, that was more pronounced than with wild-type Tpm2.2. Single ATP turnover kinetic experiments indicate that the introduced mutations have opposite effects on product release kinetics. While the hypercontractile Tpm2.2 mutants accelerated product release, the hypocontractile mutants decelerated product release from myosin, thus having either an activating or inhibitory influence on myosin motor performance, which agrees with the muscle disease phenotypes caused by these mutations.

A Novel Variant in TPM3 Causing Muscle Weakness and Concomitant Hypercontractile Phenotype.

A novel variant of unknown significance c.8A > G (p.Glu3Gly) in TPM3 was detected in two unrelated families. TPM3 encodes the transcript variant Tpm3.12 (NM_152263.4), the tropomyosin isoform specifically expressed in slow skeletal muscle fibers. The patients presented with slowly progressive muscle weakness associated with Achilles tendon contractures of early childhood onset. Histopathology revealed features consistent with a nemaline rod myopathy. Biochemical in vitro assays performed with reconstituted thin filaments revealed defects in the assembly of the thin filament and regulation of actin-myosin interactions. The substitution p.Glu3Gly increased polymerization of Tpm3.12, but did not significantly change its affinity to actin alone. Affinity of Tpm3.12 to actin in the presence of troponin ± Ca2+ was decreased by the mutation, which was due to reduced interactions with troponin. Altered molecular interactions affected Ca2+-dependent regulation of the thin filament interactions with myosin, resulting in increased Ca2+ sensitivity and decreased relaxation of the actin-activated myosin ATPase activity. The hypercontractile molecular phenotype probably explains the distal joint contractions observed in the patients, but additional research is needed to explain the relatively mild severity of the contractures. The slowly progressive muscle weakness is most likely caused by the lack of relaxation and prolonged contractions which cause muscle wasting. This work provides evidence for the pathogenicity of the TPM3 c.8A > G variant, which allows for its classification as (likely) pathogenic.

Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin.

Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca2+-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structural changes in the coiled coil caused by disease-related mutations. In this study, we designed 97-residue peptides, Tpm1.164-154 and Tpm3.1265-155, focusing on the actin-binding period 3 of two muscle isoforms. Using these peptides, we evaluated the effects of cardiomyopathy mutations: I92T and V95A in Tpm1.1, and congenital myopathy mutations R91P and R91C in Tpm3.12. We introduced a cysteine at the N-terminus of each fragment to promote the formation of the coiled-coil structure by disulfide bonds. Dimerization of the designed peptides was confirmed by gel electrophoresis in the presence and absence of dithiothreitol. Using circular dichroism, we showed that all mutations decreased coiled coil stability, with Tpm3.1265-155R91P and Tpm1.164-154I92T having the most drastic effects. Our experiments also indicated that adding the N-terminal cysteine increased coiled coil stability demonstrating that our design can serve as an effective tool in studying the coiled-coil fragments of various proteins.

Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments.

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca2+-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy-skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca2+, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca2+]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca2+]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca2+-dependent regulation of actin-myosin interactions.

Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding.

Tropomyosin (Tpm) binds along actin filaments and regulates myosin binding to control muscle contraction. Tropomodulin binds to the pointed end of a filament and regulates actin dynamics, which maintains the length of a thin filament. To define the structural determinants of these Tpm functions, we examined the effects of two congenital myopathy mutations, A4V and R91C, in the Tpm gene, TPM3, which encodes the Tpm3.12 isoform, specific for slow-twitch muscle fibers. Mutation A4V is located in the tropomodulin-binding, N-terminal region of Tpm3.12. R91C is located in the actin-binding period 3 and directly interacts with actin. The A4V and R91C mutations resulted in a 2.5-fold reduced affinity of Tpm3.12 homodimers for F-actin in the absence and presence of troponin, and a two-fold decrease in actomyosin ATPase activation in the presence of Ca2+ . Actomyosin ATPase inhibition in the absence of Ca2+ was not affected. The Ca2+ sensitivity of ATPase activity was decreased by R91C, but not by A4V. In vitro, R91C altered the ability of tropomodulin 1 (Tmod1) to inhibit actin polymerization at the pointed end of the filaments, which correlated with the reduced affinity of Tpm3.12-R91C for Tmod1. Molecular dynamics simulations of Tpm3.12 in complex with F-actin suggested that both mutations reduce the affinity of Tpm3.12 for F-actin binding by perturbing the van der Waals energy, which may be attributable to two different molecular mechanisms-a reduced flexibility of Tpm3.12-R91C and an increased flexibility of Tpm3.12-A4V.

The primary cause of muscle disfunction associated with substitutions E240K and R244G in tropomyosin is aberrant behavior of tropomyosin and response of actin and myosin during ATPase cycle.

Using the polarized photometry technique we have studied the effects of two amino acid replacements, E240K and R244G, in tropomyosin (Tpm1.1) on the position of Tpm1.1 on troponin-free actin filaments and the spatial arrangement of actin monomers and myosin heads at various mimicked stages of the ATPase cycle in the ghost muscle fibres. E240 and R244 are located in the C-terminal, seventh actin-binding period, in f and b positions of the coiled-coil heptapeptide repeat. Actin, Tpm1.1, and myosin subfragment-1 (S1) were fluorescently labeled: 1.5-IAEDANS was attached to actin and S1, 5-IAF was bound to Tpm1.1. The labeled proteins were incorporated in the ghost muscle fibres and changes in polarized fluorescence during the ATPase cycle have been measured. It was found that during the ATPase cycle both mutant tropomyosins occupied a position close to the inner domain of actin. The relative amount of the myosin heads in the strongly-bound conformations and of the switched on actin monomers increased at mimicking different stages of the ATPase cycle. This might be one of the reasons for muscle dysfunction in congenital fibre type disproportion caused by the substitutions E240K and R244G in tropomyosin.

Deviations in conformational rearrangements of thin filaments and myosin caused by the Ala155Thr substitution in hydrophobic core of tropomyosin.

Effects of the Ala155Thr substitution in hydrophobic core of tropomyosin Tpm1.1 on conformational rearrangements of the components of the contractile system (Tpm1.1, actin and myosin heads) were studied by polarized fluorimetry technique at different stages of the actomyosin ATPase cycle. The proteins were labelled by fluorescent probes and incorporated into ghost muscle fibres. The substitution violated the blocked and closed states of thin filaments stimulating abnormal displacement of tropomyosin to the inner domains of actin, switching actin on and increasing the relative number of the myosin heads in strong-binding state. Furthermore, the mutant tropomyosin disrupted the major function of troponin to alter the distribution of the different functional states of thin filaments. At low Ca2+ troponin did not effectively switch thin filament off and the myosin head lost the ability to drive the spatial arrangement of the mutant tropomyosin. The information about tropomyosin flexibility obtained from the fluorescent probes at Cys190 indicates that this tropomyosin is generally more rigid, that obviously prevents tropomyosin to bend and adopt the appropriate conformation required for proper regulation.

Cofilin - a protein controlling dynamics of actin filaments.

Cofilins are evolutionary conserved proteins present in all Eukaryotic cells. Their primary function is dynamic reorganization of actin cytoskeleton. Two cofilin isoforms are known: cofilin 1, present in all studied non-muscle cells and in embryonic muscle cells, and cofilin 2, which dominates in mature skeletal and cardiac muscles. Polypeptide chains of both isoforms fold into a structure homological to a conservative ADF (actin depolymerizing factor) domain, which is characteristic of actin depolymerizing factor. In cofilin molecule two actin-binding sites were found. One site binds monomeric and filamentous actin, the second one interacts only with the filament. Binding of cofilin to actin filament causes a change in the orientation of subunits, which results in filament severing. This increases number of ends which can either elongate or shorten the filament, depending on the conditions. Cofilin interactions with monomeric actin decreases availability of polymerization-competent actin subunits. Cofilin activity is controlled by phosphorylation, binding membrane phospholipids, local pH and oxidative stress. Under conditions of oxidative stress oxidation of cysteine residues leads to formation of dimers, which are able to cross-link actin filaments. Stable actin-cofilin rods save cellular ATP, which is not used during active polymerization process. This facilitates faster cell recovery from the stress. The final cellular reaction on the environmental stimuli is a resultant of cofilin activity and activities of other actin-binding proteins, which function either synergistically or antagonistically. Due to the central role in the regulation of actin filaments dynamics, cofilin is involved in development of cancer, neurodegenerative diseases, congenital myopathies and cardiomyopathies.

Molecular mechanisms of deregulation of the thin filament associated with the R167H and K168E substitutions in tropomyosin Tpm1.1.

Point mutations R167H and K168E in tropomyosin Tpm1.1 (TM) disturb Ca2+-dependent regulation of the actomyosin ATPase. To understand mechanisms of this defect we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres using the polarized fluorescence microscopy. It was found that both mutations disturbed the mode of troponin operation in the fibres. At high Ca2+, troponin increased the fraction of actin monomers that were in the "switched on" state, but both mutant tropomyosins were shifted toward the outer actin domains, which decreased the fraction of strongly bound myosin heads throughout the ATPase cycle. At low Ca2+, the R167H-TM was located close to the outer actin domains, which reduced the number of strongly-bound myosin heads. However, under these conditions troponin increased the number of actin monomers that were switched on. The K168E-TM was displaced far to the outer actin domains and troponin binding decreased the fraction of switched on actin monomers, but the proportion of the strongly bound myosin heads was abnormally high. Thus, the mutations differently disturbed transmission of conformational changes between troponin, tropomyosin and actin, which is essential for the Са2+-dependent regulation of the thin filament.

Abnormal movement of tropomyosin and response of myosin heads and actin during the ATPase cycle caused by the Arg167His, Arg167Gly and Lys168Glu mutations in TPM1 gene.

Amino acid substitutions: Arg167His, Arg167Gly and Lys168Glu, located in a consensus actin-binding site of the striated muscle tropomyosin Tpm1.1 (TM), were used to investigate mechanisms of the thin filament regulation. The azimuthal movement of TM strands on the actin filament and the responses of the myosin heads and actin subunits during the ATPase cycle were studied using fluorescence polarization of muscle fibres. The recombinant wild-type and mutant TMs labelled with 5-IAF, 1,5-IAEDANS-labelled S1and FITC-phalloidin F-actin were incorporated into the ghost muscle fibres to acquire information on the orientation of the probes relative to the fibre axis. The substitutions Arg167Gly and Lys168Glu shifted TM strands into the actin filament centre, whereas Arg167His moved TM towards the periphery of the filament. In the presence of Arg167Gly-TM and Lys168Glu-TM the fraction of actin monomers that were switched on and the number of the myosin heads strongly bound to F-actin were abnormally high even under conditions close to relaxation. In contrast, Arg167His-TM decreased the fraction of switched on actin and reduced the formation of strongly bound myosin heads throughout the ATPase cycle. We concluded that the altered TM-actin contacts destabilized the thin filament and affected the actin-myosin interactions.

Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament.

Tropomyosin and troponin are bound to the actin filament to control the contraction of striated muscle in the Ca-dependent manner. The interactions between both regulatory proteins important for the regulation process are not fully understood. To gain more insight into the mechanisms of the thin filament regulation by skeletal α-tropomyosin and troponin, we analyzed effects of seven myopathy-related substitutions: Leu99Met, Ala155Thr, Arg167Gly, Arg167Cys, Arg167His, Lys168Glu, and Arg244Gly. All substitutions reduced Ca-dependent activation of the actomyosin ATPase. The effects of mutations in Arg167 and Lys168 were the most severe. The amino acid substitutions did not significantly affect troponin binding to the whole filament, but reduced 1.2-2.8 fold the affinity of troponin to tropomyosin alone. The excimer fluorescence of N-(1-pyrene)iodoacetamide, a probe attached to the central Cys190, demonstrated that substitutions located near the troponin core domain-binding region strongly affected conformational changes accompanying the tropomyosin-troponin interactions. The thermal stability of all tropomyosin mutants was lower than the stability of the wild type tropomyosin, with TM reduced by 5.3-8.5°C. Together the analyses demonstrated that the myopathy-causing mutations affected tropomyosin structure and led to changes in interactions between tropomyosin and troponin, which impaired the transition of the thin filament from the inactive off to the active on state.

CacyBP/SIP as a novel modulator of the thin filament.

The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

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.

[Calcium ions in the regulation of acto-myosin interactions]. / Udzial jonów wapnia w regulacji oddzialywan aktyny z miozyna.

Muscle contraction and different forms of motility of nonmuscle cells depend on cyclic interactions between actin filaments and myosin motors. Calcium ions are the main intracellular signal, which induces activation of actin-myosin interactions. Depending on the cell type and the class of myosin, the molecular mechanisms of regulation are different and take place on two levels - actin filament and myosin. In striated muscle, the actin thin filament is regulated by the troponin-tropomyosin complex. In smooth muscle and nonmuscle cells, actin filaments are predominantly regulated by caldesmon. The control of myosin activity in these cells also depends on the myosin light chain phosphorylation and the phosphorylation of the heavy chain. Direct binding of calcium ions to the myosin light chains (which could be calmodulin molecules) was observed in myosin from molluscan muscle and in some unconventional myosins. Intensive world-wide studies allow us to understand details of the mechanisms of actin-myosin interactions. In this article, we present the contemporary view on these mechanisms.

Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end-to-end overlaps.

Tropomyosins are dimeric rod-like proteins which polymerize along actin filaments and regulate interactions with other actin-binding proteins. Homologous sequences responsible for the binding of tropomyosin to consecutive actin monomers repeat along tropomyosin and are called actin-binding periods. In this work, the localization of tropomyosin isoforms on actin alone and on actin­myosin complex was evaluated by measuring Förster resonance energy transfer (FRET) distances between a donor (AEDANS) attached to either the N-terminal actin-binding period 1 or to the central actin-binding period 5 and an acceptor (DABMI) bound to actin's Cys374. The recombinant -tropomyosin isoforms­TM2, TM5a, and TM1b9a, used in this study, had various amino acid sequences of the N- and C-termini forming the end-to-end overlap. Although the sequences of actin-binding period 5 of the three isoforms were identical, the donor­acceptor distances calculated for each isoform varied between 38.6 and 41.5 Å. Differences in FRET distances between the three tropomyosin isoforms labeled in actin-binding period 1 varied between 34.8 and 40.2 Å. Rigor binding of myosin heads to actin increased all measured distances. The degree and cooperativity of myosin-induced shift was different for each of the isoforms and actin-binding periods. The structural differences correlate with cooperative regulation of actin-activated S1 ATPase by the three tropomyosins. The results indicate that amino acid sequences of the end-to-end overlap determine specific orientation of tropomyosin isoform on actin. This can be important for steric and cooperative regulation of the actin filament and determine functional specificity of multiple tropomyosin isoforms present in eucaryotic cells.

[Role of tropomyosin isoforms in diversification of actin filaments functions]. / Rola izoform tropomiozyny w róznicowaniu funkcji filamentów aktynowych.

Actin interactions with myosin as well as dynamic polymerization and depolymerization of actin filaments underlie various forms of cellular motility. Both mechanisms are under control of regulatory proteins, among which tropomyosins take a central position. Tropomyosin isoforms can be tissue-specific or constitutive, expressed in different cell types. Tropomyosin expression pattern depends on the cell type and developmental stage. Several tropomyosin isoforms can simultanously co-exist in a single cell, and they are subjected to sorting to different cellular compartments. Due to specific localization, tropomyosins determine interactions of the filaments with actin binding proteins, and ascertain various filaments functions.

Role of actin C-terminus in regulation of striated muscle thin filament.

In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca(2+) and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca(2+), but it reduces tropomyosin-troponin affinity in the absence of Ca(2+). In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca(2+)-dependent regulation of the ATPase activity is preserved. Without Ca(2+) the ATPase activity is fully inhibited, but in the presence of Ca(2+) the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation.

The DNase-I binding loop of actin may play a role in the regulation of actin-myosin interaction by tropomyosin/troponin.

Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation. Possible participation of loop 38-52 in this communication has recently been questioned based on unimpaired Ca(2+) regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met(47) and Gly(48). We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly(42) and Val(43), on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N- and C-terminal parts of loop 38-52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38-52 of actin in communication between tropomyosin and myosin through changes in actin structure.

Structural determinants of cooperativity in acto-myosin interactions.

Regulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtained with various tropomyosin isoforms and mutants help us to understand better the structural requirements in the thin filament for cooperative protein interactions. Forming an end-to-end overlap between neighboring tropomyosin molecules is not necessary for the cooperativity of the thin filament activation. When direct contacts between tropomyosin molecules are disrupted, the conformational changes in the filament are most probably transmitted cooperatively through actin subunits, although the exact nature of these changes is not known. The function of tropomyosin ends, alternatively expressed in various isoforms, is to confer specific actin affinity. Tropomyosin's affinity or actin is directly related to the size of the apparent cooperative unit defined as the number of actin subunits turned into the active state by binding of one myosin head. Inner sequences of tropomyosin, particularly actin-binding periods 3 to 5, play crucial role in myosin-induced activation of the thin filament. A plausible mechanism of tropomyosin function in this process is that inner tropomyosin regions are either specifically recognized by myosin or they define the right actin conformation required for tropomyosin movement from its blocking position.