High Affinity Binding of Brain Myosin-Va to F-actin Induced by Calcium in the Presence of ATP

Abstract

Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca2+ in a highly cooperative manner. We demonstrate here that Ca2+ also induces myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca2+-induced cosedimentation. The Ca 2+-induced binding was inhibited by low temperature or by 0.6 M NaCl, but not by 1% Triton X-100. Tight binding between myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca2+ was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actomyosin-Va under Ca2+-induced binding conditions showed tightly packed F-actin bundles cross-linked by myosin-Va. Our data demonstrate that high affinity binding of myosin-Va and F-actin in the presence of ATP or 5′-O-(thiotriphosphate) is induced by micromolar concentrations of Ca 2+. Since Ca2+ regulates both the actin binding properties and actin-activated ATPase of myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of myosin Va.