Powerstroke

Binding of myosin to actin triggers pivoting of the myosin head and shortening of the sarcomere. This is the powerstroke. 

Fig. 31. Implications about the contractile mechanism in insect flight muscle. Blue is insect flight muscle SI shape in pre-powerstroke state (Al-Khayat et al., 2003), and green is chicken skeletal muscle SI in the rigor state with no nucleotide bound (Rayment et al., 1993a). The actin filament (right) is shown with the Z-band at the bottom and M-band at the top. A transition from the pre-powerstroke/resting SI shape to the rigor/end of post-powerstroke shape would involve an axial swing of the lever arm by 100 A, resulting in the sliding of the actin filaments past the myosin filaments and toward the M-band.

Al-Khayat, H. A., Hudson, L., Reedy, M. K., Irving, T. C., and Squire, J. M. (2003). Myosin head configuration in relaxed insect flight muscle X-ray modeled resting crossbridges in a pre-powerstroke state are poised for actin binding. Biophys. J. 85, 1063-1079. 

B. Actin Binding Appears to Drive the Powerstroke via a /J-Sheet Twist.. 188... 

To date, three primary conformations of the myosin crossbridge that can be associated with states in the Lymn-Taylor cycle have been identified. These have been named the post-rigor structure (Fig. 2 and state 2 in Fig. 1), the pre-powerstroke structure (corresponding to the myosin products complex, M.D.P , state 3 in Fig. 1), and the rigor Iihe (or rigor structure if it is associated with actin) state (shown as state 1 in Fig. 1). A comparison of these structures leads to the identification the following important conformationally flexible elements ... 

Fig. 3. Details of the seven-stranded /1-sheet and associated structures (A and B) in the post-rigor conformation and (C and D) in the pre-powerstroke conformation. The orientation of A and C is at right angles to that shown in Fig. 2. When attached to actin, it corresponds to that shown in Fig. 5B. The colors are as in Fig. 2. The views shown in B and D are at right angles to A and C looking out radially from the axis of the actin helix. Note the kink in the relay helix shown in C and D that leads to a 60° rotation of the converter domain. This in turn rotates the lever arm 60°. The P-loop (which constitutes the ATP-binding site) and the adjoining a-helix are shown in yellow. The flanking switch sequences (1 and 2) are also shown. The strands of the /1-sheet are numbered from the N-terminal (distal) end of the sheet. The lower part of strand 5 (light blue) constitutes switch 2. In the post-rigor state, switch 2 lies out of the plane of the /1-sheet (open) and in the pre-powerstroke state switch 2 is in the plane of the /1-sheet (closed).

It is important to note that the binding of ATP places the /P, into the pocket formed by SW1 and the P-loop, which promotes the moving in of SW2. The effects of this movement are twofold the relay helix is kinked and thereby re-primed, and the enzymatically active site is formed. In the pre-powerstroke state, the nucleotide is completely enclosed by the positioning of SW1 and SW2. This conformation appears to be the enzymatically active form of myosin. Moreover, this is the preferred stable structure of myosin in the presence of nucleotide. 

The position of the converter domain depends on whether the relay helix has a kink near its middle point. The kink in the relay helix occurs in the pre-powerstroke state. The kink leads to a rotation of the converter domain through 60°. Removing the kink causes the lever arm to rotate back by 60°, which is the elementary structural event in the powerstroke. Conversely, creating the kink is the priming action necessary to reach the start of the powerstroke. 

When compared with post-rigor or pre-powerstroke states the structural effects of cleft closure appear to include the movement of SW1, which opens the nucleotide-binding pocket, together with a twist of the central /Lsheet, which is associated with a large movement of the P-loop that considerably modifies the nucleotide binding site. Partial closure of the actin-binding cleft and a very similar twisting of the /3-sheet were also seen in the nucleotide-free structure of Dictyostelium myosin II reported by Reubold et al. (2003). The myosin V atomic model can be fitted without deformation into the electron microscope three-dimensional (3D) reconstruction of decorated actin (Holmes et al., 2004). For this and other... 

Fig. 4. The actin-binding cleft between the upper (red) and lower (gray) 50K domains (orientation as in Fig. 5A). In A (rigor-like), the cleft is shut. In B (pre-powerstroke), the outer end of the cleft (that forms the actin-binding site) is fully open, but the apex or inner end of the cleft (next to the nucleotide-binding pocket ATP is shown in B) is closed. This closure is brought about by the switch 2 element (SW2) being in the closed conformation. In C (post-rigor), both the outer end and the inner end are open. SW2 is open. In A and B the dispositions of SW2 are similar, but not identical. We refer to them as closed 1 (Cj) and closed 2 (C2), respectively.

Using the fit of myosin V into the cryoelectron microscope density as the basis of the rigor complex, by adding back the missing lever arm one can generate the model of the end-of-powerstroke actin-myosin complex shown in Fig. 5 (Holmes et al, 2004). 

Myosin therefore appears to possess two mechanisms for relieving the kink in the relay helix either by moving out the lower 50K domain (SW2 closed to SW2 open) or by twisting the /1-sheet, which gives the distal end of the relay helix more space. The first mechanism (in the reverse order, SW2 open to SW2 closed) occurs in the absence of actin after ATP binds in the nucleotide pocket. This is the recovery stroke leading to the priming of the powerstroke. However, when the myosin is bound to actin, it appears that only the second mechanism is relevant (indeed, it would be rather... 

Fig. 6. The /Tsheet can twist. A comparison of the pre-powerstroke state (A) with the rigor-like state (B). For this comparison, strands 6 and 7 of the /i-sheet (shown edge on) have been aligned (this corresponds closely with an alignment of the lower 50K domains). The SW2 elements (light blue, below strand 5) in the two states are in very similar positions, as is the proximal part of the relay helix (light blue). In A, the outer part of the relay helix (dark blue) is kinked. In B, the relay helix is straight. A comparison of A and B shows that the distal end of the /l-sheet (strands 1-4) twists through 7-8°. Note that this leads to a displacement of the P-loop.

The strongly bound pre-powerstroke state or top-of-powerstroke state is the transitory state labeled 4 in Fig. 1. It is experimentally difficult to characterize this either kinetically or structurally. At present, the structure can only be guessed at by an extrapolation of the properties of the adjoining structures. It seems very likely that the actin-binding cleft closes on strong binding in the top-of-powerstroke state. Comparison of the structures of the pre-powerstroke and post-rigor states with the nucleotide-free... 

Fig. 7. The strongly bound top-of-powerstroke state. Shown is the truncated myosin crossbridge without the lever arm. The orientation is as in Fig. 5A. (A) Pre-powerstroke state with the upper 50K domain shown in yellow. (B) The rigor-like state with the upper 50K domain of the pre-powerstroke state (yellow) superimposed on the upper 50K domain of the rigor-like state (red). (C) The model produced by taking the superimposed orientation of the upper 50K domain and combining it with the original pre-powerstroke coordinates. This generates a pre-powerstroke state with a shut actin-binding cleft that serves as a model of the ephemeral strongly bound top-of-powerstroke state.

Fig. 8. Switch 1 (SW1) opens on strong binding to actin. The orientation as in Fig. 5B, with the lower 50K domain and the lever arm omitted. (A) The weakly attached pre-powerstroke crossbridge. (B) Model of the strongly attached top-of-powerstroke structure (for details see text). Note that the SW1 element has been pulled away from the environment of the phosphates by strong binding to actin.

Fig. 9. The powerstroke. (A) A model of the strongly bound pre-powerstroke state.

The rest of the nucleotide binding site, P-loop and SW2, are intact. However, the subsequent twisting of the /(-sheet during the powerstroke moves the P-loop and considerably modifies the nucleotide-binding site. 

The modeled structure may also be used to generate the attached top-of-powerstroke state. This is shown in Fig. 9A compared with the rigor conformation (Fig. 9B). The same geometry for the attachment to actin has been used, as was found by electron microscopy for the binding of myosin V to actin. The lever arm from chicken skeletal myosin has been used to complete the model. 

Goureux et al. (2004) discuss the differences among rigor-like, postrigor, and pre-powerstroke states in detail. Their conclusions are based on the crystallographic structures of myosin V with and without bound nucleotide. We use a somewhat simplified scheme to describe the differences and similarities among the three conformers of myosin that we have identified as being represented in the Lymn-Taylor cycle. These are summarized in Table I. 

The apparent positions in the Lymn-Taylor cycle of the structural states that we refer to as the pre-powerstroke, rigor-like, and post-powerstroke conformations are shown in Fig. 10. Thus it appears that three of the four... 

The fourth actin-bound, top-of-powerstroke state is ephemeral. In the original Lymn—Taylor model it is not clear if this fourth state is strongly or weakly bound to actin, but P release, ADP release, and force generation all occur during the transition 4 to 1. Thus, in the following discussion, we attempt to break this transition down into a series of elementary events and to explore if it is possible to order the biochemical and mechanical events and to correlate them with structural changes. 

We first review the biochemical evidence for the events from state 1 to 3 (rigor to pre-powerstroke) before considering the events associated with 3 to 1 (pre-powerstroke to rigor). 

The events associated with SW2 closure followed by ATP hydrolysis have been described above. The idea originally explored in 1999 (Geeves and Holmes, 1999), that SW2 must close onto the yP of ATP before ATP hydrolysis takes place, has been elegantly demonstrated for both Dic-tyostelium myosin II and rabbit fast muscle myosin II (Malnasi-Gsizmadia et al, 2001 Urbanke and Wray, 2001). The kinetic and spectroscopic studies are all compatible with the post-rigor and pre-powerstroke crystal structures. 

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