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 have found that this chimeric molecule is highly processive in single molecule in vitro motility assays and has a step-size larger that would be predicted for a molecule with only 2IQ motifs. Rotary shadowing electron microscopy reveals that the length of the neck is extended by the added SAH domain. This suggests that the SAH domain engineered into the chimera is possibly acting as an extension of the lever arm or at least makes a flexible tether that allows the free head to explore more of the actin filament in searching for a binding site. We have carried out optical trapping nanometry of a myosin X construct in which a leucine zipper was included at the end of the SAH domain to force dimerization of the molecule. The length of the powerstroke is consistent with the concept that the SAH domain is acting as a portion of the lever arm. We find that at low trap stiffness this molecule takes multiple steps in the optical trap.

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National Heart, Lung, and Blood Institute
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Baboolal, Thomas G; Sakamoto, Takeshi; Forgacs, Eva et al. (2009) The SAH domain extends the functional length of the myosin lever. Proc Natl Acad Sci U S A 106:22193-8