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Flexibility within the heads of muscle myosin-2 molecules.

ABSTRACT: 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.

SUBMITTER: Billington N

PROVIDER: S-EPMC3919154 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.

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The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation.

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Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.

Project description:Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles.

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Project description:The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca2+-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

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Compliance Accelerates Relaxation in Muscle by Allowing Myosin Heads to Move Relative to Actin.

Project description:The mechanisms that limit the speed at which striated muscles relax are poorly understood. This work presents, to our knowledge, novel simulations that show that the time course of relaxation is accelerated by interfilamentary movement resulting from series compliance; force drops faster when myosin heads move relative to actin during relaxation. This insight was obtained by using cross-bridge distribution techniques to simulate the mechanical behavior of half-sarcomeres that were connected in series with springs of varying stiffness. (The springs mimic the combined effects of half-sarcomere heterogeneity and muscle's series elastic component.) Half-sarcomeres that shortened by >?10 nm when they were activated subsequently relaxed with a biphasic profile; force initially declined slowly and approximately linearly before collapsing with a fast exponential time course. Stretches imposed during the linear phase quickened relaxation, while shortening movements prolonged the time course. These predictions are consistent with data from experiments performed by many other groups using single muscle fibers and isolated myofibrils. When half-sarcomeres were linked to stiff springs (so that they did not shorten appreciably during the simulations), force relaxed with a slow exponential time course and did not show biphasic behavior. Together, these results suggest that fast relaxation of striated muscle is an emergent property that reflects multiscale interactions within the muscle architecture. The nonlinear behavior during relaxation reflects perturbations to the dynamic coupling of regulated binding sites and cycling myosin heads that are induced by interfilamentary movement.

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Insights into Actin-Myosin Interactions within Muscle from 3D Electron Microscopy.

Project description:Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy (cryoEM) of F-actin, decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3D imaging by electron microscopy (EM) that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large water bug Lethocerus sp. The Lethocerus flight muscle possesses a particularly favorable filament arrangement that enables all the myosin cross-bridges contacting the actin filament to be visualized in a thin section. This review covers the history of this effort and the progress toward visualizing the complex set of conformational changes that myosin heads make when binding to actin in several static states, as well as the fast frozen actively contracting muscle. The efforts have revealed a consistent pattern of changes to the myosin head structures as determined by X-ray crystallography needed to explain the structure of the different actomyosin interactions observed in situ.

| S-EPMC6479483 | biostudies-literature

The mesa trail and the interacting heads motif of myosin II.

Project description:Myosin II molecules in the thick filaments of striated muscle form a structure in which the heads interact with each other and fold back onto the tail. This structure, the "interacting heads motif" (IHM), provides a mechanistic basis for the auto-inhibition of myosin in relaxed thick filaments. Similar IHM interactions occur in single myosin molecules of smooth and nonmuscle cells in the switched-off state. In addition to the interaction between the two heads, which inhibits their activity, the IHM also contains an interaction between the motor domain of one head and the initial part (subfragment 2, S2) of the tail. This is thought to be a crucial anchoring interaction that holds the IHM in place on the thick filament. S2 appears to cross the head at a specific location within a broader region of the motor domain known as the myosin mesa. Here, we show that the positive and negative charge distribution in this part of the mesa is complementary to the charge distribution on S2. We have designated this the "mesa trail" owing to its linear path across the mesa. We studied the structural sequence alignment, the location of charged residues on the surface of myosin head atomic models, and the distribution of surface charge potential along the mesa trail in different types of myosin II and in different species. The charge distribution in both the mesa trail and the adjacent S2 is relatively conserved. This suggests a common basis for IHM formation across different myosin IIs, dependent on attraction between complementary charged patches on S2 and the myosin head. Conservation from mammals to insects suggests that the mesa trail/S2 interaction plays a key role in the inhibitory function of the IHM.

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