Myosin X is an unconventional myosin that has been implicated in filopodial development in mammals. We have recently characterized its steady-state and transient state MgATPase activity. Myosin X contains a region of predicted coiled-coil 120 residues long. However, the highly charged nature, and pattern of charges in the proximal 36-residues, appears incompatible with coiled-coil formation. Circular dichroism, NMR and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single a-helix (SAH domain) in solution and does not dimerize to form coiled-coil, even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin X containing the motor, the three calmodulin binding domains and the full-length predicted coiled-coil showed that it was mostly monomeric at physiological protein concentration. In dimers, the molecules were only joined at their extreme distal ends and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin V HMM obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus, the predicted coiled-coil of myosin X forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins VI, VIIa, and myoM, and could function to increase the size of the working stroke.? ? We have engineered a chimeric myosin in which the motor domain and first two IQ regions of myosin V are fused with the predicted SAH domain of myoM followed by the coiled-coil rod of myosin V. We plan to use this chimerical protein to test whether the molecule is processive and, if so, what is its step size. In addition, we will negative stain electron microscopy to determine the length of the neck region. These studies are in progess.
| Knight, Peter J; Thirumurugan, Kavitha; Xu, Yuhui et al. (2005) The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head. J Biol Chem 280:34702-8 |
| Murphy, Patrick J M; Morishima, Yoshihiro; Kovacs, Jeffrey J et al. (2005) Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone. J Biol Chem 280:33792-9 |
| Kovacs, Mihaly; Wang, Fei; Sellers, James R (2005) Mechanism of action of myosin X, a membrane-associated molecular motor. J Biol Chem 280:15071-83 |
