N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

Neurofilament Light Protein Rod Domain Exhibits Structural Heterogeneity.

Neurofilaments are neuron-specific proteins that belong to the intermediate filament (IFs) protein family, with the neurofilament light chain protein (NFL) being the most abundant. The IFs structure typically includes a central coiled-coil rod domain comprised of coils 1A, 1B, and 2, separated by linker regions. The thermal stability of the IF molecule plays a crucial role in its ability for self-association. In the current study, we investigated the thermal stability of NFL coiled-coil domains by analyzing a set of recombinant domains and their fusions (NFL1B, NFL1A+1B, NFL2, NFL1B+2, and NFLROD) via circular dichroism spectroscopy and differential scanning calorimetry. The thermal stability of coiled-coil domains is evident in a wide range of temperatures, and thermal transition values (Tm) correspond well between isolated coiled-coil domains and full-length NFL. NFL1B has a Tm of 39.4 °C, and its' fusions, NFL1A+1B and NFL1B+2, have a Tm of 41.9 °C and 41.5 °C, respectively. However, in the case of NFL2, thermal denaturation includes at least two thermal transitions at 37.2 °C and 62.7 °C. These data indicate that the continuous α-helical structure of the coil 2 domain has parts with varied thermal stability. Among all the NFL fragments, only NFL2 underwent irreversible heat-induced denaturation. Together, these results unveil the origin of full-length NFL's thermal transitions, and reveal its domains structure and properties.

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.

Structural and Functional Properties of Tropomyosin Isoforms Tpm4.1 and Tpm2.1.

Tropomyosin (Tpm) is one of the most important partners of actin filament that largely determines its properties. In animal organisms, there are different isoforms of Tpm, which are believed to be involved in the regulation of various cellular functions. However, molecular mechanisms by which various Tpm cytoplasmic regulate of the functioning of actin filaments are still poorly understood. Here, we investigated the properties of Tpm2.1 and Tpm4.1 isoforms and compared them to each other and to more extensively studied Tpm isoforms. Tpm2.1 and Tpm4.1 were very similar in their affinity to F-actin, thermal stability, and resistance to limited proteolysis by trypsin, but differed markedly in the viscosity of their solutions and thermal stability of their complexes with F-actin. The main difference of Tpm2.1 and Tpm4.1 from other Tpm isoforms (e.g., Tpm1.6 and Tpm1.7) was their extremely low thermal stability as measured by the CD and DSC methods. We suggested the possible causes of this instability based on comparing the amino acid sequences of Tpm4.1 and Tpm2.1 with the sequences of Tpm1.6 and Tpm1.7 isoforms, respectively, that have similar exon structure.

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.

Effect of Neurodegenerative Mutations in the NEFL Gene on Thermal Denaturation of the Neurofilament Light Chain Protein.

Effects of E90K, N98S, and A149V mutations in the light chain of neurofilaments (NFL) on the structure and thermal denaturation of the NFL molecule were investigated. By using circular dichroism spectroscopy, it was shown that these mutations did not lead to the changes in α-helical structure of NFL, but they caused noticeable effects on the stability of the molecule. We also identified calorimetric domains in the NFL structure by using differential scanning calorimetry. It was shown that the E90K replacement leads to the disappearance of the low-temperature thermal transition (domain 1). The mutations cause changes in the enthalpy of NFL domains melting, as well as lead to the significant changes in the melting temperatures (Tm) of some calorimetric domains. Thus, despite the fact that all these mutations are associated with the development of Charcot-Marie-Tooth neuropathy, and two of them are even located very close to each other in the coil 1A, they affect differently structure and stability of the NFL molecule.

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.

Pseudo-phosphorylation of essential light chains affects the functioning of skeletal muscle myosin.

The work aimed to investigate how the phosphorylation of the myosin essential light chain of fast skeletal myosin (LC1) affects the functional properties of the myosin molecule. Using mass-spectrometry, we revealed phosphorylated peptides of LC1 in myosin from different fast skeletal muscles. Mutations S193D and T65D that mimic natural phosphorylation of LC1 were produced, and their effects on functional properties of the entire myosin molecule and isolated myosin head (S1) were studied. We have shown that T65D mutation drastically decreased the sliding velocity of thin filaments in an in vitro motility assay and strongly increased the duration of actin-myosin interaction in optical trap experiments. These effects of T65D mutation in LC1 observed only with the whole myosin but not with S1 were prevented by double T65D/S193D mutation. The T65D and T65D/S193D mutations increased actin-activated ATPase activity of S1 and decreased ADP affinity for the actin-S1 complex. The results indicate that pseudo-phosphorylation of LC1 differently affects the properties of the whole myosin molecule and its isolated head. Also, the results show that phosphorylation of LC1 of skeletal myosin could be one more mechanism of regulation of actin-myosin interaction that needs further investigation.

Properties of Cardiac Myosin with Cardiomyopathic Mutations in Essential Light Chains.

The effects of cardiomyopathic mutations E56G, M149V, and E177G in the MYL3 gene encoding essential light chain of human ventricular myosin (ELCv), on the functional properties of cardiac myosin and its isolated head (myosin subfragment 1, S1) were investigated. Only the M149V mutation upregulated the actin-activated ATPase activity of S1. All mutations significantly increased the Ca2+-sensitivity of the sliding velocity of thin filaments on the surface with immobilized myosin in the in vitro motility assay, while mutations E56G and M149V (but not E177G) reduced the sliding velocity of regulated thin filaments and F-actin filaments almost twice. Therefore, despite the fact that all studied mutations in ELCv are involved in the development of hypertrophic cardiomyopathy, the mechanisms of their influence on the actin-myosin interaction are different.

Impact of Troponin in Cardiomyopathy Development Caused by Mutations in Tropomyosin.

Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin-myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners.

De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), caused by mutations in thin filament proteins, manifests as moderate cardiac hypertrophy and is associated with sudden cardiac death (SCD). We identified a new de novo variant, c.656A>T (p.D219V), in the TPM1 gene encoding cardiac tropomyosin 1.1 (Tpm) in a young SCD victim with post-mortem-diagnosed HCM. We produced recombinant D219V Tpm1.1 and studied its structural and functional properties using various biochemical and biophysical methods. The D219V mutation did not affect the Tpm affinity for F-actin but increased the thermal stability of the Tpm molecule and Tpm-F-actin complex. The D219V mutation significantly increased the Ca2+ sensitivity of the sliding velocity of thin filaments over cardiac myosin in an in vitro motility assay and impaired the inhibition of the filament sliding at low Ca2+ concentration. The molecular dynamics (MD) simulation provided insight into a possible molecular mechanism of the effect of the mutation that is most likely a cause of the weakening of the Tpm interaction with actin in the "closed" state and so makes it an easier transition to the "open" state. The changes in the Ca2+ regulation of the actin-myosin interaction characteristic of genetic HCM suggest that the mutation is likely pathogenic.

The effects of the tropomyosin cardiomyopathy mutations on the calcium regulation of actin-myosin interaction in the atrium and ventricle differ.

The molecular mechanisms of pathogenesis of atrial myopathy associated with hypertrophic (HCM) and dilated (DCM) mutations of sarcomeric proteins are still poorly understood. For this, one needs to investigate the effects of the mutations on actin-myosin interaction in the atria separately from ventricles. We compared the impact of the HCM and DCM mutations of tropomyosin (Tpm) on the calcium regulation of the thin filament interaction with atrial and ventricular myosin using an in vitro motility assay. We found that the mutations differently affect the calcium regulation of actin-myosin interaction in the atria and ventricles. The DCM E40K Tpm mutation significantly reduced the maximum sliding velocity of thin filaments with ventricular myosin and its Ca2+-sensitivity. With atrial myosin, its effects were less pronounced. The HCM I172T mutation reduced the Ca2+-sensitivity of the sliding velocity of filaments with ventricular myosin but increased it with the atrial one. The HCM L185R mutation did not affect actin-myosin interaction in the atria. The results indicate that the difference in the effects of Tpm mutations on the actin-myosin interaction in the atria and ventricles may be responsible for the difference in pathological changes in the atrial and ventricular myocardium.

Comparative structural and functional studies of low molecular weight tropomyosin isoforms, the TPM3 gene products.

Tropomyosin (Tpm) is an actin-associated protein and key regulator of actin filament structure and dynamics in muscle and non-muscle cells where it participates in many vital processes. Human non-muscle cells produce many Tpm isoforms; however, little is known yet about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five low molecular weight Tpm isoforms (Tpm3.1, Tpm3.2, Tpm3.4, Tpm3.5, and Tpm3.7), the products of TPM3 gene, which significantly differ by alternatively spliced internal exon 6 (6a or 6b) and C-terminal exon 9 (9a, 9c or 9d). Our results clearly demonstrate that the properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. These differences can be important in further studies to explain why these Tpm isoforms play a key role in organization and dynamics of the cytoskeleton.

Acidosis modifies effects of phosphorylated tropomyosin on the actin-myosin interaction in the myocardium.

Phosphorylation of α-tropomyosin (Tpm1.1), a predominant Tpm isoform in the myocardium, is one of the regulatory mechanisms of the heart contractility. The Tpm 1.1 molecule has one site of phosphorylation, Ser283. The degree of the Tpm phosphorylation decreases with age and also changes in heart pathologies. Myocardial pathologies, in particular ischemia, are usually accompanied by pH lowering in the cardiomyocyte cytosol. We studied the effects of acidosis on the structural and functional properties of the pseudo-phosphorylated form of Tpm1.1 with the S283D substitution. We found that in acidosis, the interaction of the N- and C-ends of the S283D Tpm molecules decreases, whereas that of WT Tpm does not change. The pH lowering increased thermostability of the complex of F-actin with S283D Tpm to a greater extent than with WT Tpm. Using an in vitro motility assay with NEM- modified myosin as a load, we assessed the effect of the Tpm pseudo-phosphorylation on the force of the actin-myosin interaction. In acidosis, the force generated by myosin in the interaction with thin filaments containing S283D Tpm was higher than with those containing WT Tpm. Also, the pseudo-phosphorylation increased the myosin ability to resist a load. We conclude that ischemia changes the effect of the phosphorylated Tpm on the contractile function of the myocardium.

Effects of myopathy-causing mutations R91P and R245G in the TPM3 gene on structural and functional properties of slow skeletal muscle tropomyosin.

Tropomyosin (Tpm) is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Numerous point mutations in the TPM3 gene encoding Tpm of slow skeletal muscles (Tpm 3.12 or γ-Tpm) are associated with the genesis of various congenital myopathies. Two of these mutations, R91P and R245G, are associated with congenital fiber-type disproportion (CFTD) characterized by hypotonia and generalized muscle weakness. We applied various methods to investigate how these mutations affect the structural and functional properties of γγ-Tpm homodimers. The results show that both these mutations lead to strong structural changes in the γγ-Tpm molecule and significantly impaired its functional properties. These changes in the Tpm properties caused by R91P and R245G mutations give insight into the molecular mechanism of the CFTD development and the weakness of slow skeletal muscles observed in this inherited disease.

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.

Mechanisms of disturbance of the contractile function of slow skeletal muscles induced by myopathic mutations in the tropomyosin TPM3 gene.

Several congenital myopathies of slow skeletal muscles are associated with mutations in the tropomyosin (Tpm) TPM3 gene. Tropomyosin is an actin-binding protein that plays a crucial role in the regulation of muscle contraction. Two Tpm isoforms, γ (Tpm3.12) and ß (Tpm2.2) are expressed in human slow skeletal muscles forming γγ-homodimers and γß-heterodimers of Tpm molecules. We applied various methods to investigate how myopathy-causing mutations M9R, E151A, and K169E in the Tpm γ-chain modify the structure-functional properties of Tpm dimers, and how this affects the muscle functioning. The results show that the features of γγ-Tpm and γß-Tpm with substitutions in the Tpm γ-chain vary significantly. The characteristics of the γγ-Tpm depend on whether these mutations located in only one or both γ-chains. The mechanism of the development of nemaline myopathy associated with the M9R mutation was revealed. At the molecular level, a cause-and-effect relationship has been established for the development of myopathy by the K169E mutation. Also, we described the structure-functional properties of the Tpm dimers with the E151A mutation, which explain muscle weakness linked to this substitution. The results demonstrate a diversity of the molecular mechanisms of myopathy pathogenesis induced by studied Tpm mutations.

Unique functional properties of slow skeletal muscle tropomyosin.

Tropomyosin (Tpm) is an α-helical coiled-coil actin-binding protein playing an essential role in the regulation of muscle contraction. The α- (Tpm 1.1) and γ- (Tpm 3.12) Tpm isoforms are expressed in fast and slow human skeletal muscles, respectively, while ß-Tpm (Tpm 2.2) is expressed in both muscle types. This results in the formation of Tpm αα- and γγ-homodimers as well as αß- and γß-heterodimers. The properties of αα-homodimer are well studied, whereas very little is known about the functional properties of γγ-homodimer and γß-heterodimer. We investigated interaction characteristics of Tpm γγ-homodimer and γß-heterodimer with actin filaments and Ca2+-regulation of actin-myosin interaction on myosin from fast and slow skeletal muscles. The results showed that complexes formed by γγ-Tpm and γß-Tpm with F-actin are more stable than those with αα-Tpm and αß-Tpm. The maximum sliding speed of regulated thin filaments with either γγ-Tpm or γß-Tpm moving over skeletal myosin was significantly less than that of the filaments with αα-Tpm or αß-Tpm. The results indicate that isoforms of Tpm along with isoforms of myosin determine of functional properties of skeletal muscles and support an idea on the combined expression of myosin and Tpm isoforms.

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.