Introduction to Muscle Contraction, Part 2

ADP.BeFx can produce both open and closed states

ADP.BeFx is thought to be an analogue for ATP. Fisher et al (Fisher et al. 1995) solved the structure of dictyostelium S1 truncated at 761 with ADP.BeFx bound in the active site and found it to be remarkably similar to chicken S1 without nucleotide. This result appears to show that the structure of the ATP state is open, which is puzzling since it would not be able to hydrolyze the ATP. Moreover, the attitude of the converter domain (and hence the neck) is close to rigor which is also unexpected for the ATP state (c.f. Figs. 6 and 4). More recently Schlichting et al (Schlichting et al. 1997) have solved the structure of an ADP.BeFx complex of truncated S1 and find it to be essentially identical to the ADP.vanadate complex.

The active site is closed and the converter domain is in the rotated configuration. The construct used in this case was truncated at position 754 and is therefore 7 residues shorter than that used by Fisher et al. This results in a tighter binding of ADP (Kurzawa et al. 1997). Apparently, on account of this difference, in the shorter construct the binding energy of ADP.BeFx is adequate to tip the scales for the closed structure, whereas in the longer construct it was not. Therefore one can picture the transition between the two forms of myosin as being sensitively poised (but well determined - intermediate states have yet to show up): the structure solved by Fisher et al apparently corresponds to the ADP-bound state whereas the structure solved by Schlichting et al corresponds to the ATP-bound state. Comparing the Fisher et al ADP.BeFx state (Fig 6) with chicken rigor (Fig 4) there is in fact a 10° movement of the lever arm. This may reflect small changes in the angle of the lever arm induced by the binding of ADP but it could also reflect species differences.

Phosphate release

Actin binds to the open form of the 50k upper / lower cleft and thereby facilitates phosphate release.

The closed structure found with the ADP.vanadate generates a tight hydrogen bonding pattern for the γ-phosphate which probably explains the high phosphate affinity. This interaction in turn is important for stabilizing the closed form. Opening the cleft destroys the γ-phosphate binding pocket. Energy-filter cryo electron microscopy of decorated actin (Schröder et al. 1997) shows that the cleft may be open in the actin-myosin complex. Therefore it seems very likely that actin binding opens the cleft rather than closes the cleft as was suggested earlier (Rayment et al. 1993). Opening the cleft destroys the phosphate binding site and facilitates γ-phosphate release by a different route to that by which it entered (a "back door enzyme" - Yount et al. 1995).

Although kinetic studies provide evidence that the actin myosin binding in the presence of nucleotide is a multi-step process, there is no structural data on an initial weak binding of the closed form to actin. A consistent scheme may be developed by postulating that there is an additional transitory state, a bent closed form. We suppose that actin binds myosin with one main set of contacts at approximately constant geometry, namely as is seen in the rigor actin-myosin complex (i.e. the open form of myosin). The 50K lower domain probably forms the invariant contacts to actin: the switch from weak to strong binding probably involves the recruitment of loops (the 50K- 20K loop and the 404 loop) from the 50K upper domain to form the strong binding state. When confronted with myosin in the closed form actin probably binds the 50K lower domain first, which binds actin weakly. The subsequent binding of the loops produces an open form which releases the γ-phosphate and binds actin strongly.

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