Precise Binding of Tropomyosin on Actin Involves Sequence-Dependent Variance in Coiled-Coil Twisting.

Often considered an archetypal dimeric coiled coil, tropomyosin nonetheless exhibits distinctive "noncanonical" core residues located at the hydrophobic interface between its component α-helices. Notably, a charged aspartate, D137, takes the place of nonpolar residues otherwise present. Much speculation has been offered to rationalize potential local coiled-coil instability stemming from D137 and its effect on regulatory transitions of tropomyosin over actin filaments. Although experimental approaches such as electron cryomicroscopy reconstruction are optimal for defining average tropomyosin positions on actin filaments, to date, these methods have not captured the dynamics of tropomyosin residues clustered around position 137 or elsewhere. In contrast, computational biochemistry, involving molecular dynamics simulation, is a compelling choice to extend the understanding of local and global tropomyosin behavior on actin filaments at high resolution. Here, we report on molecular dynamics simulation of actin-free and actin-associated tropomyosin, showing noncanonical residue D137 as a locus for tropomyosin twist variation, with marked effects on actin-tropomyosin interactions. We conclude that D137-sponsored coiled-coil twisting is likely to optimize electrostatic side-chain contacts between tropomyosin and actin on the assembled thin filament, while offsetting disparities between tropomyosin pseudorepeat and actin subunit periodicities. We find that D137 has only minor local effects on tropomyosin coiled-coil flexibility, (i.e., on its flexural mobility). Indeed, D137-associated overtwisting may actually augment tropomyosin stiffness on actin filaments. Accordingly, such twisting-induced stiffness of tropomyosin is expected to enhance cooperative regulatory translocation of the tropomyosin cable over actin.

Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function.

Elongated tropomyosin, associated with actin-subunits along the surface of thin filaments, makes electrostatic interactions with clusters of conserved residues, K326, K328, and R147, on actin. The association is weak, permitting low-energy cost regulatory movement of tropomyosin across the filament during muscle activation. Interestingly, acidic D292 on actin, also evolutionarily conserved, lies adjacent to the three-residue cluster of basic amino acids and thus may moderate the combined local positive charge, diminishing tropomyosin-actin interaction and facilitating regulatory-switching. Indeed, charge neutralization of D292 is connected to muscle hypotonia in individuals with D292V actin mutations and linked to congenital fiber-type disproportion. Here, the D292V mutation may predispose tropomyosin-actin positioning to a myosin-blocking state, aberrantly favoring muscle relaxation, thus mimicking the low-Ca2+ effect of troponin even in activated muscles. To test this hypothesis, interaction energetics and in vitro function of wild-type and D292V filaments were measured. Energy landscapes based on F-actin-tropomyosin models show the mutation localizes tropomyosin in a blocked-state position on actin defined by a deeper energy minimum, consistent with augmented steric-interference of actin-myosin binding. In addition, whereas myosin-dependent motility of troponin/tropomyosin-free D292V F-actin is normal, motility is dramatically inhibited after addition of tropomyosin to the mutant actin. Thus, D292V-induced blocked-state stabilization appears to disrupt the delicately poised energy balance governing thin filament regulation. Our results validate the premise that stereospecific but necessarily weak binding of tropomyosin to F-actin is required for effective thin filament function.

Spontaneous transitions of actin-bound tropomyosin toward blocked and closed states.

After muscle contraction, myosin cross-bridge heads detach from thin actin filaments during relaxation. Structural and kinetic data of cross-bridge-thin filament interactions have shown that tropomyosin's position on F-actin is biased toward the blocked or closed states when myosin detaches. It is not clear if structural linkages between tropomyosin and myosin cross-bridge heads, or tropomyosin and Ca2+-free troponin, drive the process or whether tropomyosin movement is energetically independent of myosin and troponin influence. Here we provide in silico data about tropomyosin dynamics on troponin/myosin-free F-actin indicating that tropomyosin moves from the open state toward blocked- or closed-state positions on actin. To follow transitions inherent to tropomyosin itself on F-actin, we performed MD simulations initiated from the blocked-, open-, and intermediate-state models and followed tropomyosin over the surface of F-actin in the absence of myosin and troponin. These MD simulations maintain tropomyosin in a cable-like conformation, including the tropomyosin overlap domain, while allowing tropomyosin to retain most of its motional freedom. Tropomyosin shows considerable azimuthal movement away from the open state toward the surrounds of a more energetically favorable blocked B-state position over F-actin. In contrast, little movement away from the B-state location is observed. Our results are consistent with previous predictions based on electrostatic interaction energy landscapes determined by rigid-body translocation of tropomyosin. They support the view that in the absence of myosin, i.e., when myosin cross-bridges detach from actin, the blocked- or closed-state positions of tropomyosin are energetically favored, while the open state is not.

Periodic trends and easy estimation of relative stabilities in 11-vertex nido-p-block-heteroboranes and -borates.

Density functional theory computations were carried out for 11-vertex nido-p-block-hetero(carba)boranes and -borates containing silicon, germanium, tin, arsenic, antimony, sulfur, selenium and tellurium heteroatoms. A set of quantitative values called "estimated energy penalties" was derived by comparing the energies of two reference structures that differ with respect to one structural feature only. These energy penalties behave additively, i.e., they allow us to reproduce the DFT-computed relative stabilities of 11-vertex nido-heteroboranes in general with good accuracy and to predict the thermodynamic stabilities of unknown structures easily. Energy penalties for neighboring heteroatoms (HetHet and HetHet') decrease down the group and increase along the period (indirectly proportional to covalent radii). Energy penalties for a five- rather than four-coordinate heteroatom, [Het(5k)(1) and Het(5k)(2)], generally, increase down group 14 but decrease down group 16, while there are mixed trends for group 15 heteroatoms. The sum of HetHet' energy penalties results in different but easily predictable open-face heteroatom positions in the thermodynamically most stable mixed heterocarbaboranes and -borates with more than two heteroatoms.

Structural increments for 11-vertex nido-phospha- and aza(carba)boranes and -borates; dependence of energy penalties on the extent of Electron Localization.

Relevant structural features and corresponding energy penalties were determined that allow to easily estimate the relative stabilities of 11-vertex nido-phospha- and aza-substituted boranes, borates, carbaboranes, and carbaborates. For this purpose, density functional theory computations at the B3LYP/6-311+G(d,p)//B3LYP/6-31G(d)+ZPE level were carried out to determine the relative energies of 95 phospha- and 46 aza(carba)boranes and -borates. Energy penalties assigned to disfavoring structural features show additive behavior and excellent precision with respect to the computed results, as in the case of 6- and 11-vertex nido-carboranes and -borates. An unsubstituted phosphorus atom was found to possess energy penalties quite similar to those of the three-electron-donating H-C group. A bare nitrogen atom has energy penalties much larger than those of a bare phosphorus atom. Four-electron-donating RP and RN moieties, however, have even more adverse energy penalties. The disfavoring effects of heteroatoms in a borane cluster are determined by the amount of electron localization, that is, primarily by the number of skeletal electrons that formally originate from the heterogroup and secondarily by the electronegativity. Heteroatom energy penalties are independent of the type of the other heteroatoms present in the same cluster. Some novel phospha(carba)borane geometries with bare and exo-substituted phosphorus atoms in the same cluster have favorable thermodynamic stabilities competitive with those of known isomers.

Structural increment system for 11-vertex nido-boranes and carboranes.

An increment system forming a set of quantitative rules that govern the relative stabilities of 11-vertex nido-boranes and carboranes is presented. Density functional theory computations at the B3LYP/6-311+G//B3LYP/6-31G level with ZPE corrections were carried out for 61 different boron hydride and carborane structures from [B(11)H(14)](-) to C(4)B(7)H(11) to determine their relative stabilities. Disfavored structural features that destabilize a cluster structure relative to a hypothetical ideal situation were identified and weighted by so-called energy penalties. The latter show additive behavior and allow us to reproduce (within 5 kcal mol(-)(1)) the DFT computed relative energies. Energy penalties for four structural features, i.e., adjacent carbon atoms, CC, a hydrogen atom bridging between a carbon and a boron atom, CH-B, an endo-terminal hydrogen atom at an open face carbon atom, CH(2) and an endo-H between two carbon atoms, C(BH(2))C for the 11-vertex nido-cluster are quite similar to those reported for the 6-vertex nido-cluster, thus showing a behavior independent of the cluster size. Hydrogen structural features, however, vary strongly with the cluster size. Two unknown 11-vertex nido-carboranes were identified which are thermodynamically more stable than known positional isomers.