William Lehman

Regulation in molluscan muscles

In molluscan muscles the factors which regulate contraction by interacting with calcium are associated with myosin. Purified myosin preparations from the striated and smooth muscles of Aequipecten and from the smooth red adductor muscle of Mercenaria bind calcium with a great affinity and the ATPase activity of this myosin combined with purified actin requires calcium. The actin-containing thin filaments of these muscles do not bind calcium and although they activate the ATPase of rabbit myosin this activity is not calcium dependent. The thin filaments of molluscs, however, combine readily in vitro with the relaxing proteins of rabbit and behave, then, like rabbit preparations. The components responsible for calcium binding and for the calcium dependence of ATPase cannot readily be removed from molluscan myosin and are not obtained from molluscan muscles or actomyosins by procedures successfully applied to rabbit preparations. Tropomyosin does not appear to be necessary for the regulation of molluscan actomyosin by calcium. It is likely that in molluscs the calcium-binding component interacts directly with myosin to prevent cross-bridge formation. The possibility that in vertebrate muscles cross-bridge formation may also be controlled by a direct interaction of some of the regulatory proteins with-myosin is discussed.

Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.

Structural basis for the activation of muscle contraction by troponin and tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca(2+) on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca(2+) and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca(2+) allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca(2+)-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca(2+), troponin operates as an inhibitor, while in activated muscles at high Ca(2+), it acts as a promoter to initiate contraction.

An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation

Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.

Tropomyosin Position on F-Actin Revealed by EM Reconstruction and Computational Chemistry

Electron microscopy and fiber diffraction studies of reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin filaments, free of constraints imposed by troponin or other actin-binding proteins, cannot be formulated, and thus optimal interfacial contacts between actin and tropomyosin remain unknown. Here, a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions, and pseudo-rotations of tropomyosin on F-actin was performed. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic residues on the successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into electron microscopy reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the seven broad and narrow motifs that typify tropomyosins twisting superhelical coiled-coil to the wide and tapering tropomyosin densities seen in surface views of F-actin-tropomyosin reconstructions. The functional implications of the F-actin-tropomyosin models determined in this work are discussed.

Gestalt-binding of tropomyosin to actin filaments

We argue that the overall behavior of tropomyosin on F-actin cannot be easily discerned by examining thin filaments reduced to their smallest interacting units. In isolation, the individual interactions of actin and tropomyosin, by themselves, are too weak to account for the specificity of the system. Instead the association of tropomyosin on actin can only be fully explained after considering the concerted action of the entire acto-tropomyosin system. We propose that the low Ka describing tropomyosin:actin interaction, when taken together with the form-fitting complementarity of tropomyosin strands on F-actin and the tendency for tropomyosin to polymerize end-to-end, make possible unique thin filament functions both locally and at higher levels of filament organization.

The Shape and Flexibility of Tropomyosin Coiled Coils: Implications for Actin Filament Assembly and Regulation

Wrapped superhelically around actin filaments, the coiled-coil alpha-helices of tropomyosin regulate muscle contraction by cooperatively blocking or exposing myosin-binding sites on actin. In non-muscle cells, tropomyosin additionally controls access of actin-binding proteins involved in cytoskeletal actin filament maintenance and remodeling. Tropomyosins global shape and flexibility play a key role in the assembly, maintenance, and regulatory switching of thin filaments yet remain insufficiently characterized. Here, electron microscopy and molecular dynamics simulations yielded conformations of tropomyosin closely resembling each other. The electron microscopy and simulations show that isolated tropomyosin has an average curved conformation with a design well matched to its superhelical shape on F-actin. In addition, they show that tropomyosin bends smoothly yet anisotropically about its distinctive helically curved conformation, without any signs of unfolding, chain separation, localized kinks, or joints. Previous measurements, assuming tropomyosin to be straight on average, mistakenly suggested considerable flexibility (with persistence lengths only approximately 3 times the proteins length). However, taking the curved average structure determined here as reference for the flexibility measurements yields a persistence length of approximately 12 lengths, revealing that tropomyosin actually is semirigid. Corresponding simulation of a triple mutant (A74L-A78V-A81L) with weak actin affinity shows that it lacks shape complementarity to F-actin. Thus, tropomyosins pre-shaped semirigid architecture is essential for the assembly of actin filaments. Further, we propose that once bound to thin filaments, tropomyosin will be stiff enough to act as a cooperative unit and move on actin in a concerted way between known regulatory states.

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca2+, and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593–606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH2 terminus (TnT-(1–153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1–153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1–153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1–153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca2+.

Tropomyosin and the steric mechanism of muscle regulation

Contraction in all muscles must be precisely regulated and requisite control systems must be able to adjust to changes in physiological and myopathic stimuli. In this chapter, we outline the structural evidence for a steric mechanism that governs muscle activity. The mechanism involves calcium and myosin induced changes in the position of tropomyosin along actin-based thin filaments. This process either blocks or uncovers myosin crossbridge binding sites on actin and consequently regulates crossbridge cycling on thin filaments, the sliding of thin and thick filaments and muscle shortening and force production.

The recruitment of acetylated and unacetylated tropomyosin to distinct actin polymers permits the discrete regulation of specific myosins in fission yeast.

Tropomyosin (Tm) is a conserved dimeric coiled-coil protein, which forms polymers that curl around actin filaments in order to regulate actomyosin function. Acetylation of the Tm N-terminal methionine strengthens end-to-end bonds, which enhances actin binding as well as the ability of Tm to regulate myosin motor activity in both muscle and non-muscle cells. In this study we explore the function of each Tm form within fission yeast cells. Electron microscopy and live cell imaging revealed that acetylated and unacetylated Tm associate with distinct actin structures within the cell, and that each form has a profound effect upon the shape and integrity of the polymeric actin filament. We show that, whereas Tm acetylation is required to regulate the in vivo motility of class II myosins, acetylated Tm had no effect on the motility of class I and V myosins. These findings illustrate a novel Tm-acetylation-state-dependent mechanism for regulating specific actomyosin cytoskeletal interactions.