Functional significance of the conserved residues in the flexible hinge region of the myosin motor domain

Analysis of the three-dimensional crystal structure of the Dictyosteliun my osin motor domain revealed that the myosin head is required to bend at resi dues Il-455 and Gly-457 to produce the conformation changes observed in the ternary complexes that resemble the pre- and post-hydrolysis states (Fishe r, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). Asp-454, Il-455, and G ly-457 of smooth muscle myosin were substituted by Ala, Met, and Ala, respe ctively, and the mechano-enzymatic activities were determined to study the role of these residues in myosin motor function. Whereas the basal steady-s tate Mg2+-ATPase activity of D454A was higher than that of the wild type, t he rate of the hydrolytic step is reduced similar to 2,000-fold and becomes rate-limiting. M-ATP rather than M-ADP-P is the predominant steady-state i ntermediate, and the initial P-i burst and the ATP-induced enhancement of i ntrinsic tryptophan fluorescence are absent in D454A. D454A binds actin in the absence of ATP but is not dissociated from actin by ATP. Moreover, acti n inhibits rather than activates the ATPase activity; consequently, D454A d oes not support actin translocating activity. I455M has normal actin-activa ted ATPase activity, P-i burst, and ATP-induced enhancement of intrinsic tr yptophan fluorescence, suggesting that the enzymatic properties are normal. However, the actin translocating activity was completely inhibited. This s uggests that the side chain at Ile-455 is critical for myosin motor activit y but not for relatively normal enzymatic function, which indicates an appa rent uncoupling between enzymatic activity and motile function. Although G4 57A has normal ATP-dependent actin dissociation, ATP hydrolytic step is red uced by similar to 10(5)-fold in the presence or absence of actin; conseque ntly, G457A does not have actin translocating activity. These results indic ate the importance of these conserved residues at the hinge region for norm al myosin motor function.