Actin-binding cleft

The status of the actin-binding cleft open, half open, or shut... 

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.

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.

D. Rigor State Actin Binding Closes the 50K Cleft. 171... 

The relay/converter conformation is the readout of the result of these inputs. In going from the post-rigor to pre-power states, the only signal is SW2 going from open to closed, which produces the relay kink and the converter up. To go from the pre-power to rigor states requires the /i-twist, which is triggered by actin binding and cleft closure. 

Fig. 1. Ribbon diagrams of Dictyostelium discoideum G-actin (Matsuura et al., 2000) and the Ascaris suum aMSP dimer (Bullock et al., 1996) at the same magnification. Actin consists of four subdomains that surround a nucleotide-binding cleft. The G-actin molecule is asymmetric, so that when it polymerizes, the filament it forms has a characteristic polarity and its two ends differ structurally. By contrast, MSP does not contain a nucleotide binding site and the polymerizing unit is a dimer in which the two MSP molecules are related by twofold rotational symmetry. Polymerization produces filaments composed of two helical subfilaments in which the dimers twofold axes are oriented perpendicular to the helix axis. Consequently, the MSP helices have no polarity and the subfilament ends are identical structurally (Bullock et al., 1998). Reproduced from The Journal of Cell Biology, 2000, vol. 149, pp. 7-12 by copyright permission of the Rockefeller University Press.

In the third phase, the ends of actin filaments are in a steady state with monomeric ATP-G-actin. After their incorporation into a filament, subunits slowly hydrolyze ATP and become stable ADP-F-actin (white). Note that the ATP-binding clefts of all the subunits are oriented in the same direction in F-actin. (b) Time course of the in vitro polymerization reaction (pink curve) reveals the initial lag period. If some actin filament fragments are added at the start of the reaction to act as nuclei, elongation proceeds immediately without any lag period (purple curve). 

First, profilin promotes the assembly of actin filaments by acting as a nucleotide-exchange factor. Profilin is the only actin-binding protein that allows the exchange of ATP for ADP. When G-actin is complexed with other proteins, ATP or ADP is trapped in the ATP-blndlng cleft of actin. However, because profilin binds to G-actln at a site opposite the... 

ATP-binding cleft, it can recharge ADP-actin monomers released from a filament, thereby replenishing the pool of ATP-actin (Figure 19-10). 

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