Myosin motor domain lever arm rotation is coupled to ATP hydrolysis

We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical fea ture is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor doma in is free, and then reverses the rotation to produce force while it is bou nd to actin. These mechanical steps are coupled to steps in the ATP hydroly sis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measu re changes in hydrodynamic size that result from lever arm rotation when va rious Ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by mus cle fibers, are compared to those of ATP gamma S and GTP, which do not. Mea surements are also made of conformational changes when the motor domain is bound to NDP's and PPi in the absence and presence of the phosphate analogu e orthovanadate, to determine the roles the nucleoside moieties of the nucl eotides have on lever arm rotation. The results indicate that for the subst rates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P-i complex as the s teady-state intermediate make the motor domain more compact, and only those substrates support force production.