Flexibility within the Heads of Muscle Myosin-2 Molecules

We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~ 23 pN·nm/rad² is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor–lever junction.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

Regulatory light chains modulate in vitro actin motility driven by skeletal heavy meromyosin

Phosphorylation and Ca²⁺-Mg²⁺ exchange on the regulatory light chains (RLCs) of skeletal myosin modulate muscle contraction. However, the relation between the mechanisms for the effects of phosphorylation and metal ion exchange are not clear. We propose that modulation of skeletal muscle contraction by phosphorylation of the myosin regulatory light chains (RLCs) is mediated by altered electrostatic interactions between myosin heads/necks and the negatively charged thick filament backbone. Our study, using the in vitro motility assay, showed actin motility on hydrophilic negatively charged surfaces only over the HMM with phosphorylated RLCs both in the presence and absence of Ca²⁺. In contrast, good actin motility was observed on silanized surfaces (low charge density), independent of RLC phosphorylation status but with markedly lower velocity in the presence of Ca²⁺. The data suggest that Ca²⁺-binding to, and phosphorylation of, the RLCs affect the actomyosin interaction by independent molecular mechanisms. The phosphorylation effects depend on hydrophobicity and charge density of the underlying surface. Such findings might be exploited for control of actomyosin based transportation of cargoes in lab-on-a chip applications, e.g. local and temporary stopping of actin sliding on hydrophilic areas along a nanosized track.     

Similarities and differences between frozen-hydrated, rigor acto-S1 complexes of insect flight and chicken skeletal muscles

The structure and function of myosin crossbridges in asynchronous insect flight muscle (IFM) have been elucidated in situ using multiple approaches. These include generating “atomic” models of myosin in multiple contractile states by rebuilding the crystal structure of chicken subfragment 1 (S1) to fit IFM crossbridges in lower-resolution electron microscopy tomograms and by “mapping” the functional effects of genetically substituted, isoform-specific domains, including the converter domain, in chimeric IFM myosin to sequences in the crystal structure of chicken S1.We prepared helical reconstructions (∼ 25 Å resolution) to compare the structural characteristics of nucleotide-free myosin0 S1 bound to actin (acto-S1) isolated from chicken skeletal muscle (CSk) and the flight muscles of Lethocerus (Leth) wild-type Drosophila (wt Dros) and a Drosophila chimera (IFI-EC) wherein the converter domain of the indirect flight muscle myosin isoform has been replaced by the embryonic skeletal myosin converter domain. Superimposition of the maps of the frozen-hydrated acto-S1 complexes shows that differences between CSk and IFM S1 are limited to the azimuthal curvature of the lever arm: the regulatory light-chain (RLC) region of chicken skeletal S1 bends clockwise (as seen from the pointed end of actin) while those of IFM S1 project in a straight radial direction. All the IFM S1s are essentially identical other than some variation in the azimuthal spread of density in the RLC region. This spread is most pronounced in the IFI-EC S1, consistent with proposals that the embryonic converter domain increases the compliance of the IFM lever arm affecting the function of the myosin motor. These are the first unconstrained models of IFM S1 bound to actin and the first direct comparison of the vertebrate and invertebrate skeletal myosin II classes, the latter for which, data on the structure of discrete acto-S1 complexes, are not readily available.     

Thiol reactivity as a sensor of rotation of the converter in myosin

Smooth muscle myosin has two reactive thiols located near the C-terminal region of its motor domain, the “converter”, which rotates by ∼70° upon the transition from the “nucleotide-free” state to the “pre-power stroke” state. The incorporation rates of a thiol reagent, 5-(((2-iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), into these thiols were greatly altered by adding ATP or changing the myosin conformation. Comparisons of the myosin structures in the pre-power stroke state and the nucleotide-free state explained why the reactivity of both thiols is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the rotation of the converter. Modeling of the myosin structure in the pre-power stroke state, in which the most reactive thiol, “SH1”, was selectively modified with IAEDANS, revealed that this label becomes an obstacle when the converter completely rotates toward its position in the pre-power stroke state, thus resulting in incomplete rotation of the converter. Therefore, we suggest that the limitation of the converter rotation by modification causes the as-yet unexplained phenomena of SH1-modified myosin, including the inhibition of 10S myosin formation and the losses in phosphorylation-dependent regulation of the basic and actin-activated Mg-ATPase activities of myosin.     

Crystal structure of the C1 domain of cardiac myosin binding protein-C: implications for hypertrophic cardiomyopathy

C-protein is a major component of skeletal and cardiac muscle thick filaments. Mutations in the gene encoding cardiac C-protein [cardiac myosin binding protein-C (cMyBP-C)] are one of the principal causes of hypertrophic cardiomyopathy. cMyBP-C is a string of globular domains including eight immunoglobulin-like and three fibronectin-like domains termed C0-C10. It binds to myosin and titin, and probably to actin, and may have both a structural and a regulatory role in muscle function. To help to understand the pathology of the known mutations, we have solved the structure of the immunoglobulin-like C1 domain of MyBP-C by X-ray crystallography to a resolution of 1.55 Å. Mutations associated with hypertrophic cardiomyopathy are clustered at one end towards the C-terminus, close to the important C1C2 linker, where they alter the structural integrity of this region and its interactions.     

An unstable head-rod junction may promote folding into the compact off-state conformation of regulated myosins

The N-terminal region of myosin's rod-like subfragment 2 (S2) joins the two heads of this dimeric molecule and is key to its function. Previously, a crystal structure of this predominantly coiled-coil region was determined for a short fragment (51 residues plus a leucine zipper) of the scallop striated muscle myosin isoform. In that study, the N-terminal 10-14 residues were found to be disordered. We have now determined the structure of the same scallop peptide in three additional crystal environments. In each of two of these structures, improved order has allowed visualization of the entire N-terminus in one chain of the dimeric peptide. We have also compared the melting temperatures of this scallop S2 peptide with those of analogous peptides from three other isoforms. Taken together, these experiments, along with examination of sequences, point to a diminished stability of the N-terminal region of S2 in regulated myosins, compared with those myosins whose regulation is thin filament linked. It seems plain that this isoform-specific instability promotes the off-state conformation of the heads in regulated myosins. We also discuss how myosin isoforms with varied thermal stabilities share the basic capacity to transmit force efficiently in order to produce contraction in their on states.     

Kinetic analysis of the slow skeletal myosin MHC-1 isoform from bovine masseter muscle

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.     

Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the thick filament (except near the tapered ends) is approximately 10 nm, or 16% of S2's total length, with an angular range encompassing 90 degrees axially and 120 degrees azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head-head interactions in IFM relaxed thick filaments.     

Alternative Exon 9-Encoded Relay Domains Affect More than One Communication Pathway in the Drosophila Myosin Head

We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide-binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced actomyosin dissociation rate constant (K₁k₊₂) and ADP affinity (K_AD), measured for the chimeric constructs IFI-9b and EMB-9a, compared to wild-type IFI and EMB values. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide-binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50-kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of actomyosin cross-bridge kinetics.     

Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin

Three-dimensional reconstructions of “barbed” and “blunted” arrowheads (Craig et al., 1980) show that these two forms arise from arrangement of scallop myosin subfragments (S1) that appear about 40 Å longer in the presence of the regulatory light chain than in its absence. A similar difference in apparent length is indicated by images of single myosin subfragments in partially decorated filaments. The extra mass is located at the end of the subfragment furthest from actin, and probably comprises part of the regulatory light chain as well as a segment of the myosin heavy chain. The fact that barbed arrowheads are also formed by myosin subfragments from vertebrate striated and smooth muscles implies that the homologous light chains in these myosins have locations similar to that of the scallop light chain.The scallop light chain probably does not extend into the actin-binding site on the myosin head, and is therefore unlikely to interfere physically with binding. Rather, regulation of actin-myosin interaction by light chains may involve Ca²⁺-dependent changes in the structure of a region near the head-tail junction of myosin.The reconstructions suggest locations for actin and tropomyosin relative to myosin that are similar to those proposed by Taylor & Amos (1981) and are consistent with a revised steric blocking model for regulation by tropomyosin. The identification of actin from these reconstructions is supported by images of partially decorated filaments that display the polarity of the actin helix relative to that of bound myosin subfragments.     

Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay

Interaction of myosin with actin in striated muscle is controlled by Ca²⁺ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.     

A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ∼ 50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca²⁺-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads (“free” and “blocked”) are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states (“swaying” heads). These heads would be available for immediate actin interaction upon Ca²⁺ activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca²⁺ on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.     

Nucleotide dependent intrinsic fluorescence changes of W29 and W36 in smooth muscle myosin

The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin. In this study, an MDE containing all the endogenous tryptophans except W512 (W512 KO-MDE) decreases in intrinsic fluorescence upon nucleotide binding, demonstrating that the intrinsic fluorescence enhancement of smooth muscle myosin is not solely due to W512. Candidates for the observed quench of intrinsic fluorescence in W512 KO-MDE include W29 and W36. Whereas the intrinsic fluorescence of W36-MDE is only slightly sensitive to nucleotide binding, that of W29-MDE is paradoxically both quenched and blue-shifted upon nucleotide binding. Steady-state and time-resolved experiments suggest that fluorescence intensity changes of W29 involve both excited-state and ground-state quenching mechanisms. These results have important implications for the role of the N-terminal domain (residues 1-76) in smooth muscle myosin in the molecular mechanism of muscle contraction.     

The chemical mechanism of microbioal denitrification

It is suggested that, in the conditions which exist in vivo, one head of a myosin molecule interacts with another head of the opposite molecule, inside the backbone of the thick filament. The other head lies outside and can interact with actin. This model is based on the fact that a dimer of the myosin heads exists and that there is a close correlation between the properties of the dimer and those of the thick filament diameter. In natural filaments, there are myosin molecules in excess and it is suggested that these molecules have their two heads outside the backbone.     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

Role of the tail in the regulated state of myosin 2

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.     

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-BeF_x complex (stable analog of S1¹-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-BeF_x 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.