Abstract
The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II minifilaments is dependent both on Mg2+ concentration and on the state of phosphorylation of three serine sites at the C-terminal end of the heavy chains. Previous electric birefringence experiments on minifilaments showed a large dependence of signal amplitude on the phosphorylation state and Mg2+ concentration, consistent with large changes in filament flexibility. These observations suggested that minifilament stiffness was important for function. We now report that the binding of nucleotides to dephosphorylated minifilaments at Mg2+ concentrations needed for optimal activity increases the flexibility by about 10-fold, as inferred from the birefringence signal amplitude increase. An increase in flexibility with nucleotide binding is not observed for dephosphorylated minifilaments at lower Mg2+ concentrations or for phosphorylated minifilaments at any Mg2+ concentrations examined. The relaxation times for minifilament rotations that are sensitive to the conformation myosin heads are also observed to depend on phosphorylation, Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II correlates with both changes in myosin head conformation and the ability of minifilaments to cycle between stiff and flexible conformations coupled to nucleotide binding and release.
Highlights
The actin-activated Mg2؉-ATPase activity of Acanthamoeba myosin II minifilaments is dependent both on Mg2؉ concentration and on the state of phosphorylation of three serine sites at the C-terminal end of the heavy chains
The addition of ATP to dephosphorylated minifilaments in 5 mM Mg2ϩ increased the signal amplitude to a magnitude similar to that observed at the lower concentration of Mg2ϩ in the absence of nucleotide
Structural Implications of the Change in Total Signal Amplitude—We previously reported that the electric birefringence signal amplitude of dephosphorylated Acanthamoeba myosin II minifilaments decreases significantly as the Mg2ϩ is increased from 1 to 4 mM, i.e., to the concentration range necessary for optimal expression of actin-activated Mg2ϩ-ATPase activity
Summary
The actin-activated Mg2؉-ATPase activity of Acanthamoeba myosin II minifilaments is dependent both on Mg2؉ concentration and on the state of phosphorylation of three serine sites at the C-terminal end of the heavy chains. The light chains stabilize the helical tail of S1 [3, 5], and removal of the light chains or the helical tail of S1 greatly reduces the ability of S1 to move actin filaments in an in vitro motility assay [6], but neither the light chains nor the helical tail of S1 are necessary for maximal actin-activated Mg2ϩATPase activity of S1 These data are consistent with the most recent structural model of the contractile cycle derived from x-ray crystallography of S1; conformational changes in the globular portion of S1 resulting from the binding and hydrolysis of ATP are transmitted through the ELC and generate a rotational movement of the helical tail of S1 [7]. The actin-activated Mg2ϩ-ATPase activities of S1 from both ELCregulated and RLC-regulated muscle myosins are unregulated (whereas HMM is regulated), suggesting that at least a portion of the tails is necessary for the appropriate coupling of S1 head and light chain conformations [4]
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