Structural model of weak binding actomyosin in the prepowerstroke state

Abstract

We present the first in silico model of the weak binding actomyosin in the initial powerstroke state, representing the actin binding-induced major structural changes in myosin. First, we docked an actin trimer to prepowerstroke myosin then relaxed the complex by a 100-ns long unrestrained molecular dynamics. In the first few nanoseconds, actin binding induced an extra primed myosin state, i.e. the further priming of the myosin lever by 18° coupled to a further closure of switch 2 loop. We demonstrated that actin induces the extra primed state of myosin specifically through the actin N terminus-activation loop interaction. The applied in silico methodology was validated by forming rigor structures that perfectly fitted into an experimentally determined EM map of the rigor actomyosin. Our results unveiled the role of actin in the powerstroke by presenting that actin moves the myosin lever to the extra primed state that leads to the effective lever swing.

Keywords: Actin; Enzyme Cycle; Enzyme Kinetics; Enzyme Mechanism; Molecular Dynamics; Myosin; Powerstroke; Protein Structure; Structural Model.

FIGURE 1.

Structural changes in myosin during its chemomechanical cycle. The initial state of the powerstroke is the weak actomyosin·ADP·P_i complex with an up position of the lever (prepowerstroke, highlighted with orange box). The magnitudes of the gray arrows refer to the flux of the reactions. The lengths of the black arrows represent the relative magnitudes of the rate constants of the steps of the enzyme cycle. The futile reaction pathway is represented with faded colors.

FIGURE 2.

The structure of the docked and relaxed rigor actomyosin complexes. Actomyosin structures are fitted into the electron density map (transparent gray) of _EMA·M_rigor,ch with their actin trimers (yellow-red-green) as best fits for the map. Myosin (blue) outside the EM map is colored white. The root mean square deviations of the backbones of _dockA·M_rigor,Dd and _relA·M_rigor,Dd from _EMA·M_rigor,ch are 6.0 ± 1.9 and 4.7 ± 1.7 Å, respectively. The root mean square deviations of the backbones of _dockA·M_rigor,sq and _relA·M_rigor,sq from _EMA·M_rigor,ch are 5.8 ± 1.8 and 5.5 ± 1.7 Å, respectively.

FIGURE 3.

The actin-binding interface of myosin in its different actin-binding states. A, the actin-binding myosin interface of the relaxed actomyosin complexes are shown as a colored surface representation of myosin (transparent gray). Loop 3 (blue), H-loop (green), activation loop (red), and other residues of the lower 50-kDa domain (yellow) have extended interactions with actin both in weak, _relA·M_up, and strong, _relA·M_rigor,Dd, _relA·M_rigor,sq, actin-binding states, whereas loop 4 (orange), cardiomyopathy loop (purple), and loop 2 (ice blue) of the upper 50-kDa domain have few interactions in the weak actin-binding state of myosin. Positions of the converter (light blue) and relay helix (cyan) are also highlighted in the myosin structure. B, in the weak actin-binding state (_relA·M_up) loop 2 (ice blue) interacts with Asp²⁵ of actin (green), whereas activation loop (red) of the upper relay (yellow) has interactions with the N-terminal region of actin. In the strong actin-binding state (_relA·M_rigor,Dd) the N-terminal part of actin positions between loop 2 and the activation loop, creating interactions with both. The position of the relay (cyan) is also presented.

FIGURE 4.

The extra primed state of the weak binding actomyosin complex. A, after docking and relaxation of myosin (gray) and actin (green-yellow), the _relA·M_up (red) possesses a further up-lever and a further closed switch 2 compared with _relM_up (cyan) and the overlaid PDB 1MMD down-lever motor domain (orange) structures. Arbitrary extensions of the levers are represented by red, cyan, and orange cylinders, respectively. Inset, positions of switch 2 and switch 1 in further up (red) and up (cyan) states of myosin. B, changes in the angle of the position of the lever (left panel) and the distance between switch 1 and switch 2 (right panel) throughout the molecular dynamic simulations of A·M_up (red), _relA·M_up,R520Q (blue), _relA·M_up,R562Q (green), _relA·M_{up,K622Q/K623Q} (pink), and as a control _relM_up (black). R520Q, R562Q, and K622Q/K623Q mutations interrupt the interactions of actin with activation loop, loop 3, and loop 2, respectively.

FIGURE 5.

Transmission of structural changes from actin to the relay/converter and nucleotide binding pocket. In the _relM_up structure, β-sheet (orange), switch 2 (pink), and the relay (cyan) are not connected through the wedge loop (gray) to the upper relay (yellow) and activation loop (red). As the activation loop creates interaction with the N-terminal region of actin (green) in _relA·M_up, a hydrophobic cluster (cloud) is formed between the upper relay, wedge loop, and relay regions. Wedge loop is also repositioned, creating interaction with switch 2. In _relA·M_up,R520Q, the hydrophobic cluster and the wedge loop-switch 2 interaction could not form, whereas they are still present in _relA·M_up,R562Q and _relA·M_{up,K622Q/K623Q} mutants.

FIGURE 6.

Motional correlations of switch 2 with different structural regions of the myosin motor domain. Motional correlations of switch 2 with different regions of myosin in the relaxed myosin and actomyosin structures are quantitatively represented by the red-white color code (scale bar). 0 value means no correlation and 1 is strong correlation. Representation of structural elements of myosin motor domain: converter (light blue), relay (cyan), upper relay (yellow), activation loop (red), loop 3 (blue), H-loop (green), loop 2 (ice blue), loop 4 (orange), cardiomyopathy loop (purple), and β-sheet (orange).

FIGURE 7.

Comparison of the extra primed state in myosin 2 and myosin 6 and their structural transmission pathways in the absence of actin. A, activation loop, switch 2, the relay and the converter region are highlighted from myosin (gray). Myosin 6 prepowerstroke state (PDB 2V26, blue) possesses a similar, or even more further primed lever and further closed switch 2 than _relA·M_up (red). The two myosin structures were aligned by their backbones. B, in the _relM_up structure, β-sheet (orange), switch 2 (pink), and the relay (blue) are not connected through the wedge loop (gray) with the upper relay (yellow) and activation loop (red). In the structure of myosin 6 (PDB 2V26), switch 2 creates interactions with the wedge loop, but the wedge loop has no further interactions with the upper relay region.