Expression Studies of Myosin X

肌凝蛋白X是一种非常规的肌凝蛋白，与哺乳动物丝状体发育有关。我们最近对其稳态和瞬态MgATPase活性进行了表征。Myosin X包含一个预测的120个残基长的螺旋状区域。然而，高电荷性质和近36个残基的电荷模式似乎与盘绕线圈形成不相容。圆二色性、核磁共振和分析性超离心表明，含有该区域的合成肽在溶液中形成稳定的单a-螺旋（SAH结构域），即使在毫摩尔浓度下也不会二聚化形成螺旋状。此外，含有马达、三个钙调蛋白结合结构域和全长预测线圈的重组肌球蛋白X的电镜显示，在生理蛋白浓度下，重组肌球蛋白X大部分是单体的。在二聚体中，分子仅在其极端远端连接，没有可见的盘绕尾。此外，单体和二聚体的颈长度都比钙调素结合域的数量预期的要长得多。相比之下，在相同条件下获得的myosin V HMM显微照片清楚地显示出卷曲的尾巴，颈部为预测长度。因此，预测的肌球蛋白X的卷曲形成了一种新的细长结构，其中近端区域是SAH结构域，远端区域是SAH结构域（或具有未知的扩展结构），仅在其末端二聚化。序列比较表明，在肌凝蛋白VI、VIIa和myoM的预测线圈结构域中可能存在类似的结构，并可能起增加工作行程大小的作用。

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 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.