Phenotypically selected mutations in myosin's actin binding domain demonstrate intermolecular contacts important for motor function.
Here, we biochemically characterize Dictyostelium myosin II mutants that were previously phenotypically selected following random mutagenesis and shown to lie in the actin binding domain [Patterson, B., & Spudich, J. A. (1996) Genetics 143, 801-810]. We show that the conditional loss of myosin-dependent activity in vivo, which results from the mutations E531Q, P536R, and R562L, is likely due to the loss of important contacts with actin. Purified wild-type and mutant myosin subfragments 1 (S1), expressed in Dictyostelium, are alike in binding to actin and releasing it in an ATP-dependent manner. Furthermore, the rates of ATP hydrolysis without actin are similar for the mutant and wild-type S1s. Thus, the mutations in the actin binding site have little effect on ATP binding or product release in the absence of actin. All three mutants, however, have impaired actin-activated ATPase activity, with apparent second-order rate constants for actin interactions that are 4-25-fold smaller than that of wild-type S1 at 30 degrees C. The mutations also cause defects in the ability to move actin, as measured by in vitro motility assays of full-length myosins. On the basis of motility of a mixture of wild-type and mutant myosins, there appears to be at least two classes of mutations, with the primary defect in either a weak or a strong actin binding state. In summary, the activities in vitro of myosins with mutations in the actin binding site suggest losses of important contacts with actin.
Giese
K C
KC
Departments of Biochemistry and Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
Spudich
J A
JA
D000199:Actins / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000199:Actins; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D009218:Myosins