Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle.
During the ATP hydrolysis cycle of the Dictyostelium myosin II motor domain, two conserved alpha-helices, the SH1/SH2 helix and the relay helix, rotate in a coordinated way to induce the swing motion of the converter domain. A network of hydrophobic and ionic interactions in these two helices and the converter may ensure that the motions of these helices are effectively transmitted to the converter. To examine the roles of these interactions in the ATPase-dependent converter swing, we disrupted two conserved hydrophobic linkages among them by means of a point mutation (I499A or F692A). The resulting mutations induced only limited changes in the kinetic parameters of ATP hydrolysis, except for a marked increase of basal MgATPase activity. However, the mutant myosins completely lost their in vitro and in vivo motor functions. Measurements of the intrinsic tryptophan fluorescence and the GFP-based FRET revealed that the converter domain of these mutants did not swing during steady-state ATP hydrolysis or in the presence of tightly trapped Mg.ADP.V(i), which shows that the point mutations induced the uncoupling of the converter swing and ATP hydrolysis cycle. These results highlight the importance of these hydrophobic linkages for transmitting the coordinated twist motions of the helices to the converter as well as the requirement of this converter swing for force generation.
Sasaki
Naoya
N
Center for Interdisciplinary Research, Tohoku University, Aramaki-aza-aoba, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
Ohkura
Reiko
R
Sutoh
Kazuo
K
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000409:Alanine / Q000235:genetics; D000818:Animals; D004023:Dictyostelium / Q000235:genetics / Q000502:physiology; D006868:Hydrolysis; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D024462:Myosin Type II / Q000172:deficiency / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D009218:Myosins / Q000037:antagonists & inhibitors / Q000737:chemistry; D010649:Phenylalanine / Q000235:genetics; D017354:Point Mutation; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D014638:Vanadates / Q000737:chemistry
D020409:Molecular Motor Proteins; D015800:Protozoan Proteins; D007532:Isoleucine; D014638:Vanadates; D010649:Phenylalanine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024462:Myosin Type II; D009218:Myosins; D000409:Alanine