Li Xiang-dong, Mabuchi Katsuhide, Ikebe Reiko, Ikebe Mitsuo
Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
Biochem Biophys Res Commun. 2004 Mar 12;315(3):538-45. doi: 10.1016/j.bbrc.2004.01.084.
We succeeded in expressing the recombinant full-length myosin Va (M5Full) and studied its regulation mechanism. The actin-activated ATPase activity of M5Full was significantly activated by Ca(2+), whereas the truncated myosin Va without C-terminal globular domain is not regulated by Ca(2+) and constitutively active. Sedimentation analysis showed that the sedimentation coefficient of M5Full undergoes a Ca(2+)-induced conformational transition from 14S to 11S. Electron microscopy revealed that at low ionic strength, M5Full showed an extended conformation in high Ca(2+) while it formed a folded shape in the presence of EGTA, in which the tail domain was folded back towards the head-neck region. Furthermore, we found that the motor domain of myosin Va folds back to the neck domain in Ca(2+) while the head-neck domain is more extended in EGTA. It is thought that the association of the motor domain to the neck inhibits the binding of the tail to the neck thus destabilizing a folded conformation in Ca(2+). This conformational transition is closely correlated to the actin-activated ATPase activity. These results suggest that the tail and neck domain play a role in the Ca(2+) dependent regulation of myosin Va.
我们成功表达了重组全长肌球蛋白Va（M5Full）并研究了其调控机制。M5Full的肌动蛋白激活的ATP酶活性被Ca(2+)显著激活，而没有C端球状结构域的截短型肌球蛋白Va不受Ca(2+)调控且组成型激活。沉降分析表明，M5Full的沉降系数经历了由Ca(2+)诱导的从14S到11S的构象转变。电子显微镜显示，在低离子强度下，M5Full在高Ca(2+)时呈伸展构象，而在EGTA存在时形成折叠形状，其中尾部结构域向头颈区域折叠回来。此外，我们发现肌球蛋白Va的运动结构域在Ca(2+)时向颈部结构域折叠，而头颈结构域在EGTA中更伸展。据认为，运动结构域与颈部的结合抑制了尾部与颈部的结合，从而破坏了Ca(2+)中折叠构象的稳定性。这种构象转变与肌动蛋白激活的ATP酶活性密切相关。这些结果表明，尾部和颈部结构域在肌球蛋白Va的Ca(2+)依赖性调控中起作用。
