Abstract
Analysis of the three-dimensional crystal structure of the Dictyostelium myosin motor domain revealed that the myosin head is required to bend at residues Ile-455 and Gly-457 to produce the conformation changes observed in the ternary complexes that resemble the pre- and post-hydrolysis states (Fisher, 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, Ile-455, and Gly-457 of smooth muscle myosin were substituted by Ala, Met, and Ala, respectively, and the mechano-enzymatic activities were determined to study the role of these residues in myosin motor function. Whereas the basal steady-state Mg2+-ATPase activity of D454A was higher than that of the wild type, the rate of the hydrolytic step is reduced approximately 2,000-fold and becomes rate-limiting. M-ATP rather than M-ADP-P is the predominant steady-state intermediate, and the initial Pi burst and the ATP-induced enhancement of intrinsic tryptophan fluorescence are absent in D454A. D454A binds actin in the absence of ATP but is not dissociated from actin by ATP. Moreover, actin inhibits rather than activates the ATPase activity; consequently, D454A does not support actin translocating activity. I455M has normal actin-activated ATPase activity, Pi burst, and ATP-induced enhancement of intrinsic tryptophan fluorescence, suggesting that the enzymatic properties are normal. However, the actin translocating activity was completely inhibited. This suggests that the side chain at Ile-455 is critical for myosin motor activity but not for relatively normal enzymatic function, which indicates an apparent uncoupling between enzymatic activity and motile function. Although G457A has normal ATP-dependent actin dissociation, ATP hydrolytic step is reduced by approximately 10(5)-fold in the presence or absence of actin; consequently, G457A does not have actin translocating activity. These results indicate the importance of these conserved residues at the hinge region for normal myosin motor function.
Highlights
Myosin is a motor protein that translocates actin filaments using energy from ATP hydrolysis
A key question is how ATP hydrolysis is coupled with the mechanical motor activity of myosin, which is thought to be universal among all myosin family members
The cleft that splits the 50-kDa central segment of myosin S1 extends from the nucleotide binding pocket to the actin binding interface, and it is proposed that this cleft closes after ATP hydrolysis [8, 11]
Summary
Materials—Restriction enzymes and modifying enzymes were purchased from New England Biolab (Beverly, MA). The fractions containing D454A were dialyzed against Buffer B (15 mM MgCl2, 1 mM DTT, and 20 mM Tris-HCl, pH 7.5) to precipitate endogenous myosin. Actin Co-sedimentation Assays—Purified wild type and mutant HMM at concentrations of 0.1 mg/ml were incubated with rabbit skeletal actin (0.06 mg/ml) in a buffer consisting of 20 mM Tris-HCl, 50 mM KCl, 1 mM DTT, and 2 mM MgCl2, pH 7.5, at 0 °C for 10 min and centrifuged at 270,000 ϫ g for 10 min. Steady-state ATPase Activity Assay—Actin-activated ATPase activity was measured at 25 °C in the assay mixture containing 0.05 mg/ml HMM, 0.2 mM CaCl2, 15 g/ml MLCK and 10 g/ml calmodulin, 1 mM MgCl2, 50 mM KCl, 0.2 mM ATP, and 30 mM Tris-HCl, pH 7.5, with various concentrations of F-actin. ATPase activity of HMM or acto-HMM was determined by measuring the liberated 32P as described previously [16]
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