50K domain

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.

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.

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.

The next stage involves the formation of a stereo-specific interaction via the lower 50K domain and is dominated by hydrophobic interactions as described above for step II. Formation of the initial stereo-specific interaction is quickly followed by closure of the cleft and formation of the complete actomyosin contact surface (strongly bound rigor-like actin-myosin interface), which is equivalent to step III above. This has been modeled above in Figs. 7 through 9. 

State 1. There is a weak actin-myosin attachment (through the lower 50K domain), which is in rapid equilibrium with the detached crossbridge. It is also in rapid equilibrium with State 2, in which the cleft closes (an equilibrium constant of 0.1-1.0). 

Thus the isometric case starts with the A-M.D.Pj state and the lower 50K cleft bound to actin. Closure of the cleft is fast and results in a distortion of the /(-sheet of the upper 50K domain. This tends to destabilize the P-loop, leading to Pj release, but, if the movement of the lever-arm and converter is not complete, then the whole process is not completed and at mM P concentration P, can rebind, leading to the loss of force and detachment. When the crossbridge is held isometric, all steps in Scheme S are in a quasi-stable dynamic equilibrium. 

Figure 14.15 Stmcture of the SI fragment of chicken myosin as a Richardson diagram (a) and a space-filling model (b). The two light chains are shown in magenta and yellow. The heavy chain is colored according to three proteolytic fragments produced by trypsin a 25-kDa N-terminal domain (green) a central 50-kDa fragment (red) divided by a cleft into a 50K upper and a 50K lower domain and a 20-kDa C-terminal domain (blue) that links the myosin head to the coiled-coil tail. The 50-kDa and 20-kDa domains both bind actin, while the 25-kDa domain binds ATP. [(b) Courtesy of 1. Rayment.]...

Fragment 3 (Mr 50K) possessed / structure, appeared globular in electron micrographs, and was found to bind to heparin. It was assumed to be the globular region at the end of the long arm of laminin. This site is one of the main heparin- and heparan sulfate-binding domains in laminin (Ott et al., 1982). 

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