Alexander M. Matyushenko

Structural and functional effects of two stabilizing substitutions, D137L and G126R, in the middle part of α‐tropomyosin molecule

Tropomyosin (Tm) is an α‐helical coiled‐coil protein that binds along the length of actin filament and plays an essential role in the regulation of muscle contraction. There are two highly conserved non‐canonical residues in the middle part of the Tm molecule, Asp137 and Gly126, which are thought to impart conformational instability (flexibility) to this region of Tm which is considered crucial for its regulatory functions. It was shown previously that replacement of these residues by canonical ones (Leu substitution for Asp137 and Arg substitution for Gly126) results in stabilization of the coiled‐coil in the middle of Tm and affects its regulatory function. Here we employed various methods to compare structural and functional features of Tm mutants carrying stabilizing substitutions Arg137Leu and Gly126Arg. Moreover, we for the first time analyzed the properties of Tm carrying both these substitutions within the same molecule. The results show that both substitutions similarly stabilize the Tm coiled‐coil structure, and their combined action leads to further significant stabilization of the Tm molecule. This stabilization not only enhances maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca2+ concentrations but also increases Ca2+ sensitivity of the actin–myosin interaction underlying this sliding. We propose that the effects of these substitutions on the Ca2+‐regulated actin–myosin interaction can be accounted for not only by decreased flexibility of actin‐bound Tm but also by their influence on the interactions between the middle part of Tm and certain sites of the myosin head.

Structural and Functional Effects of Cardiomyopathy-Causing Mutations in the Troponin T-Binding Region of Cardiac Tropomyosin.

Hypertrophic cardiomyopathy (HCM) is a severe heart disease caused by missense mutations in genes encoding sarcomeric proteins of cardiac muscle. Many of these mutations are identified in the gene encoding the cardiac isoform of tropomyosin (Tpm), an α-helical coiled-coil actin-binding protein that plays a key role in Ca2+-regulated contraction of cardiac muscle. We employed various methods to characterize structural and functional features of recombinant human Tpm species carrying HCM mutations that lie either within the troponin T-binding region in the C-terminal part of Tpm (E180G, E180V, and L185R) or near this region (I172T). The results of our structural studies show that all these mutations affect, although differently, the thermal stability of the C-terminal part of the Tpm molecule: mutations E180G and I172T destabilize this part of the molecule, whereas mutation E180V strongly stabilizes it. Moreover, various HCM-causing mutations have different and even opposite effects on the stability of the Tpm-actin complexes. Studies of reconstituted thin filaments in the in vitro motility assay have shown that those HCM-associated mutations that lie within the troponin T-binding region of Tpm similarly increase the Ca2+ sensitivity of the sliding velocity of the filaments and impair their relaxation properties, causing a marked increase in the sliding velocity in the absence of Ca2+, while mutation I172T decreases the Ca2+ sensitivity and has no influence on the sliding velocity under relaxing conditions. Finally, our data demonstrate that various HCM mutations can differently affect the structural and functional properties of Tpm and cause HCM by different molecular mechanisms.

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.

Transient interaction between the N-terminal extension of the essential light chain-1 and motor domain of the myosin head during the ATPase cycle

The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of myosin heads with actin filaments. Myosin head (myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the myosin head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeFx complex (stable analog of S1∗-AТP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeFx complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.

Effect of Cardiomyopathic Mutations in Tropomyosin on Calcium Regulation of the Actin—Myosin Interaction in Skeletal Muscle

Tropomyosin plays an important role in the regulation of actin—myosin interaction in striated muscles. Mutations in the tropomyosin gene disrupt actin—myosin interaction and lead to myopathies and cardiomyopathies. Tropomyosin with mutations in the α-chain is expressed in both the myocardium and skeletal muscles. We studied the effect of mutations in the α-chain of tropomyosin related to hypertrophic (D175N and E180G) and dilated cardiomyopathies (E40K and E54K) on calcium regulation of the actin–myosin interaction in skeletal muscles. We analyzed the calcium-dependent sliding velocity of reconstructed thin filaments containing F-actin, troponin, and tropomyosin over myosin surface in an in vitro motility assay. Mutations D175N and E180G in tropomyosin increased the sliding velocity and its calcium sensitivity, while mutation E40K reduced both these parameters. E54K mutation increased the sliding velocity of thin filaments, but did not affect its calcium sensitivity.

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.

The Effect of Stabilizing Mutations in the Central Part of α-Chain of Tropomyosin on the Bending Stiffness of Reconstructed Thin Filaments that Contain Its αβ-Heterodimers

We studied the effect of the replacement of two highly conserved noncanonical residues in the α-chain of tropomyosin, that is, Asp137 and Gly126, with the canonical residues, Leu and Arg, on the mechanical properties of reconstructed thin filaments that contain αβ-heterodimers of tropomyosin. For this purpose, the reconstructed thin filaments that contain fibrillar actin, tropomyosin, and troponin were stretched with an optical trap. The resulting strain–force diagrams were analyzed using a mathematical model proposed previously in order to estimate the bending stiffness. It was shown that the thin filaments that contain αβ-heterodimers of tropomyosin with α-chains of the pseudo-wild type, i.e., that contain the C190A substitution, have approximately the same bending stiffness as the filament with αα-homodimers of tropomyosin. The stabilizing substitution D137L in the α-chain of tropomyosin did not cause a statistically significant change in the bending stiffness of the filaments that contain αβ-heterodimers of tropomyosin, whereas the G126R and G126R/D137L substitutions led to a moderate increase in this stiffness. This increase in stiffness was, however, much less pronounced than that for the filaments that contain αα-homodimers of tropomyosin with these substitutions in both α-chains. The relationship between the results obtained in this study and the previously published data on the effects of these stabilizing substitutions in the α-chain of tropomyosin on the structural and functional properties of thin filaments with αβ-heterodimers of tropomyosin is discussed.

Thermal unfolding of homodimers and heterodimers of different skeletal-muscle isoforms of tropomyosin

We applied differential scanning calorimetry (DSC) to investigate the structural properties of three isoforms of tropomyosin (Tpm), α, β, and γ, expressed from different genes in human skeletal muscles. We compared specific features of the thermal unfolding of αα, ββ, and γγ Tpm homodimers, as well as of αβ and γβ Tpm heterodimers. The results show that the thermal stability of γγ homodimer is much higher than that of αα homodimer which, in turn, is much more thermostable than the ββ homodimer. The stability of the γβ Tpm heterodimer is much lower than that of the γγ homodimer, and its thermal unfolding is quite different from that for γγ and ββ homodimers, whereas the unfolding of the αβ heterodimer is roughly similar to that of the αα homodimer.