Cooperativity between two heads of Dictyostelium myosin II in in vitro motility and ATP hydrolysis

To elucidate the significance of the two-headed structure of myosin II, we have engineered and characterized recombinant single-headed myosin II. A ta il segment of a myosin II heavy chain fused with a His-tag was expressed in wild-type Dictyostelium cells. Single-headed myosin, which consists of a f ull length myosin heavy chain and a tagged tail, was isolated on the basis of the affinities for Nickel agarose and actin. Actin sliding velocity by t he single-headed myosin was about half of the two-headed, whereas the minim um density of the heads to support continuous movement was twofold higher. Actin-activated MgATPase activity of the single-headed myosin in solution i n the presence of 24 mu M actin was less than half of the two headed. This decrease is primarily because of fourfold-elevated Kapp for actin and secon dary to 40% lower Vmax, These results suggest that the two heads of a Dicty ostelium myosin II molecule act cooperatively on an actin filament. We prop ose a mechanism by which two heads move actin efficiently based on the coop erativity.