Flexibility of Acanthamoeba myosin rod minifilaments

Previous electric birefringence experiments have shown that the actin-activ ated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the abi lity of minifilaments to cycle between flexible and stiff conformations. Th e cooperative transition between conformations was shown to depend on Mg2concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction betw een the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expect ed to disrupt the alpha-helical coiled-coil structure of the rod, this regi on was anticipated to be the flexible site. We have now cloned and expresse d the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation a nd electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifi laments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabili ze the stiff conformation. Less Mg2+ is required to induce minifilament sti ffening if proline-1244 is replaced with alanine. Deleting the entire junct ion region (25 amino acids) results in a even greater decrease in the Mg2concentration necessary for the transition. The HMM-LMM junction does indee d seem to act as a Mg2+-dependent flexible hinge.