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Coiled-coil tropomyosin, localized on actin filaments in virtually all eukaryotic cells, serves as a gatekeeper regulating access of the motor protein myosin and other actin-binding proteins onto the thin filament surface. Tropomyosin’s modular pseudo-repeating pattern of approximately 39 amino acid residues is designed to allow binding of the coiled-coil to successive actin subunits along thin filaments. Even though different tropomyosin isoforms contain varying numbers of repeat modules, the pseudo-repeat length, in all cases, matches that of a single actin subunit. Thus, the seven pseudo-repeats of 42 nm long muscle tropomyosin bind to seven successive actin subunits along thin filaments, while simultaneously bending into a super-helical conformation that is preshaped to the actin filament helix. In order to form a continuous cable on thin filaments that is free of gaps, adjacent tropomyosin molecules polymerize head-to-tail by means of a short (∼9 residue) overlap. Several laboratories have engineered peptides to mimic the N- and C-terminal tropomyosin association and to characterize the overlap structure. All overlapping domains examined show a compact N-terminal coiled-coil inserting into a partially opened C-terminal partner, where the opposing coiled-coils at the overlap junction face each other at up to ∼90° twist angles. Here, Molecular Dynamics (MD) simulations were carried out to determine constraints on the formation of the tropomyosin overlap complex and to assess the amount of twisting exhibited by full-length tropomyosin when bound to actin. With the exception of the last 20–40 C- and N-terminal residues, we find that the average tropomyosin structure closely resembles a “canonical” model proposed in the classic work of McLachlan and Stewart, displaying perfectly symmetrical supercoil geometry matching the F-actin helix with an integral number of coiled-coil turns, a coiled-coil helical pitch of 137 Å, a superhelical pitch of 770 Å, and no localized pseudo-rotation. Over the middle 70% of tropomyosin, the average twisting of the coiled-coil deviates only by 10° from the canonical model and the torsional freedom is very small (std. dev. of 7°). This small degree of twisting cannot yield the orthogonal N- and C-termini configuration observed experimentally. In marked contrast, considerable coiled-coil unfolding, splaying and twisting at N- and C-terminal ends is observed, providing the conformational plasticity needed for head-to-tail nexus formation. [Copyright &y& Elsevier]

Highlights: [•] Head-to-tail linkage is required for tropomyosin cable formation on F-actin. [•] Molecular dynamics of tropomyosin overlap domains were carried out. [•] N- to C-terminal coiled-coils face each other at an orthogonal angle. [•] The bending angle of the overlap domain and the rest of the molecule are comparable. [•] This curvature ensures tropomyosin assembly as an uninterrupted cable on F-actin. [ABSTRACT FROM AUTHOR]

The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC's degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k. The resulting equilibrium constant $$ K_{\text{B}} \equiv 1/K_{\text{I}} $$ varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa <5.5) with a Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations. [ABSTRACT FROM AUTHOR]

Abstract: Point mutations targeting muscle thin filament proteins are the cause of a number of cardiomyopathies. In many cases, biological effects of the mutations are well-documented, whereas their structural and mechanical impact on filament assembly and regulatory function is lacking. In order to elucidate molecular defects leading to cardiac dysfunction, we have examined the structural mechanics of two tropomyosin mutants, E180G and D175N, which are associated with hypertrophic cardiomyopathy (HCM). Tropomyosin is an α-helical coiled-coil dimer which polymerizes end-to-end to create an elongated superhelix that wraps around F-actin filaments of muscle and non-muscle cells, thus modulating the binding of other actin-binding proteins. Here, we study how flexibility changes in the E180G and D175N mutants might affect tropomyosin binding and regulatory motion on F-actin. Electron microscopy and Molecular Dynamics simulations show that E180G and D175N mutations cause an increase in bending flexibility of tropomyosin both locally and globally. This excess flexibility is likely to increase accessibility of the myosin-binding sites on F-actin, thus destabilizing the low-Ca2+ relaxed-state of cardiac muscle. The resulting imbalance in the on–off switching mechanism of the mutants will shift the regulatory equilibrium towards Ca2+-activation of cardiac muscle, as is observed in affected muscle, accompanied by enhanced systolic activity, diastolic dysfunction, and cardiac compensations associated with HCM and heart failure. [Copyright &y& Elsevier]

Abstract: The Hill two-state cooperativity model and the McKillop–Geeves (McK–G) three-state model predict very similar binding traces of myosin subfragment 1 (S1) binding to regulated actin filaments in the presence and absence of calcium, and both fit the experimental data reasonably well [Chen et al., Biophys. J., 80, 2338–2349]. Here, we compared the Hill model and the McK–G model for binding myosin S1 to regulated actin against three sets of experimental data: the titration of regulated actin with S1 and the kinetics of S1 binding of regulated actin with either excess S1 to actin or excess actin to S1. Each data set was collected for a wide range of specified calcium concentrations. Both models were able to generate reasonable fits to the time course data and to titration data. The McK–G model can fit all three data sets with the same calcium-concentration-sensitive parameters. Only K B and K T show significant calcium dependence, and the parameters have a classic pCa curve. A unique set of the Hill model parameters was extremely difficult to estimate from the best fits of multiple sets of data. In summary, the McK–G cooperativity model more uniquely resolves parameters estimated from kinetic and titration data than the Hill model, predicts a sigmoidal dependence of key parameters with calcium concentration, and is simpler and more suitable for practical use. [Copyright &y& Elsevier]

Abstract: The inherent flexibility of rod-like tropomyosin coiled-coils is a significant factor that constrains tropomyosin’s complex positional dynamics on actin filaments. Flexibility of elongated straight molecules typically is assessed by persistence length, a measure of lengthwise thermal bending fluctuations. However, if a molecule’s equilibrium conformation is curved, this formulation yields an “apparent” persistence length (∼100nm for tropomyosin), measuring deviations from idealized straight conformations which then overestimate actual dynamic flexibility. To obtain the “dynamic” persistence length, a true measurement of flexural stiffness, the average curvature of the molecule must be taken into account. Different methods used in our studies for measuring the dynamic persistence length directly from Molecular Dynamics (MD) simulations of tropomyosin are described here in detail. The dynamic persistence length found, 460±40nm, is ∼12-times longer than tropomyosin and 5-times the apparent persistence length, showing that tropomyosin is considerably stiffer than previously thought. The longitudinal twisting behavior of tropomyosin during MD shows that the amplitude of end-to-end twisting fluctuation is ∼30° when tropomyosin adopts its near-average conformation. The measured bending and twisting flexibilities are used to evaluate different models of tropomyosin motion on F-actin. [Copyright &y& Elsevier]

Summary: Wrapped superhelically around actin filaments, the coiled-coil α-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. Tropomyosin''s 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 ∼3 times the protein''s length). However, taking the curved average structure determined here as reference for the flexibility measurements yields a persistence length of ∼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, tropomyosin''s 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. [Copyright &y& Elsevier]

The ends of coiled-coil tropomyosin molecules are joined together by nine to ten residue-long head-to-tail “overlapping domains”. These short four-chained interconnections ensure formation of continuous tropomyosin cables that wrap around actin filaments. Molecular Dynamics simulations indicate that the curvature and bending flexibility at the overlap is 10–20% greater than over the rest of the molecule, which might affect head-to-tail filament assembly on F-actin. Since the penultimate residue of striated muscle tropomyosin, Ser283, is a natural target of phosphorylating enzymes, we have assessed here if phosphorylation adjusts the mechanical properties of the tropomyosin overlap domain. MD simulations show that phosphorylation straightens the overlap to match the curvature of the remainder of tropomyosin while stiffening it to equal or exceed the rigidity of canonical coiled-coil regions. Corresponding EM data on phosphomimetic tropomyosin S283D corroborate these findings. The phosphorylation-induced change in mechanical properties of tropomyosin likely results from electrostatic interactions between C-terminal phosphoSer283 and N-terminal Lys12 in the four-chain overlap bundle, while promoting stronger interactions among surrounding residues and thus facilitating tropomyosin cable assembly. The stiffening effect of D283-tropomyosin noted correlates with previously observed enhanced actin–tropomyosin activation of myosin S1-ATPase, suggesting a role for the tropomyosin phosphorylation in potentiating muscle contraction. [ABSTRACT FROM AUTHOR]

large number of tropomyosin (Tm) isoforms function as gatekeepers of the actin filament, controlling the spatiotemporal access of actin-binding proteins to specialized actin networks. Residues ~40-80 vary significantly among Tm isoforms, but the impact of sequence variation on Tm structure and interactions with actin is poorly understood, because structural studies have focused on skeletal muscle Tmα. We describe structures of N-terminal fragments of smooth muscle Tmα and Tmβ (sm-Tmα and sm-Tmβ). The 2.0-Å structure of sm-Tmα81 (81-aa) resembles that of skeletalTm α, displaying a similar super-helical twist matching the contours of the actin filament. The 1.8-Å structure of sm-Tmα98 (98-aa) unexpectedly reveals an antiparallel coiled coil, with the two chainsstaggeredbyonly4aminoacidsanddisplayinghydrophobic core interactions similar to those of the parallel dimer. In contrast, the 2.5-Å structure of sm-Tmβ98, containing Gly-Ala-Ser at theN terminus to mimic acetylation, reveals a parallel coiled coil. None of the structures contains coiled-coil stabilizing elements, favoring the formation of head-to-tail overlap complexes in four of five crystallographically independent parallel dimers. These complexes show similarly arranged 4-helix bundles stabilized by hydrophobic interactions, but the extent of the overlap varies between sm-Tmβ98 and sm-Tmα81 from 2 to 3 helical turns. The formation of overlap complexes thus appears to be an intrinsic property of theTmcoiled coil, with the specific nature of hydrophobic contacts determining the extent of the overlap. Overall, there sults suggest that sequence variation among Tm isoforms has a limited effect on actin binding but could determine its gatekeeper function. [ABSTRACT FROM AUTHOR]