Ca2+-induced activation of ATPase activity of myosin Va is accompanied with a large conformational change

• Published in: Biochemical and biophysical research communications 2004-03, Vol.315 (3), p.538-545
• Main Authors: Li, Xiang-dong, Mabuchi, Katsuhide, Ikebe, Reiko, Ikebe, Mitsuo
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Adenosine Triphosphatases - metabolism; Animals; Baculoviridae - genetics; Calcium - chemistry; Calcium - metabolism; Calmodulin - metabolism; Cell Line; Enzyme Activation - drug effects; Mice; Microscopy, Electron; Models, Molecular; Muscle, Skeletal - chemistry; Myosin Heavy Chains - chemistry; Myosin Heavy Chains - genetics; Myosin Heavy Chains - metabolism; Myosin Heavy Chains - ultrastructure; Myosin Type V - chemistry; Myosin Type V - genetics; Myosin Type V - metabolism; Myosin Type V - ultrastructure; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Potassium Chloride - pharmacology; Protein Conformation; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - ultrastructure; Spodoptera - virology; Ultracentrifugation - methods; Xenopus
• ISSN: 0006-291X
• 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.