Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (P i ) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantumclassical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HP γ O 4 2− state, but in the H 2 P γ O 4 − state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (P γ O 3 − ) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.T he molecular motor myosin cyclically interacts with the actin filament to generate the mechanical force that is used in living cells to achieve muscle contraction (1), cytokinesis (2, 3), and intracellular cargo transport (4). Hydrolysis of one ATP molecule per cycle provides the free energy that drives the actomyosin interaction cycle, as originally described by Lymn and Taylor (5). ATP is the common energy currency in biology, and is extremely stable in aqueous solution (6, 7). ATPases, the enzymes that catalyze the hydrolysis of the P β -O-P γ anhydride linkage in ATP, are ubiquitous in biology because they are needed to accelerate the release of free energy stored in ATP. Myosin manages to speed up the hydrolysis by a factor of 10 7 over the uncatalyzed rate in solution. The experimental uncatalyzed energy barrier is 29 kcal mol −1 (7,8), and the catalyzed barrier has been determined experimentally between 14.4 kcal mol −1 (9) and 14.8 kcal mol −1 (10). Understanding how myosin achieves its ATPase function is necessary to understand how myosin works as a motor, but also helps one to understand the functioning of the multitude of other nucleotide hydrolyzing enzymes. The catalytic mechanism of ATP hydrolysis in myosin has been studied extensively with methods such as protein crystallography (11)(12)(13)(14), mutagenesis (15, 16), photochemical kinet...

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.

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.

ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites.

Background: It is unknown how enzymes actually achieve the catalytic pathways proposed for ATP hydrolysis. Results: A quantum mechanical analysis of myosin ATPase quantifies the relevant interactions that stabilize a metaphosphate intermediate. Conclusion:The protein is designed to stabilize this metaphosphate, key to the enzymatic mechanism. Significance: This yields a chemically consistent model for the catalytic strategy of nucleotide-hydrolyzing enzymes in general.

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.

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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-Ca 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.