D-loop of actin differently regulates the motor function of myosins II and V.
To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. On the other hand, single molecules of myosin V "walked" with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the actomyosin-ADP.P(i) state of both myosins.
Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku,Tokyo, Japan.
Hiroaki
H
Kubota
Sergey V
SV
Mikhailenko
Harumi
H
Okabe
Hideki
H
Taguchi
Shin'ichi
S
Ishiwata
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D024462:Myosin Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024701:Myosin Type V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011817:Rabbits
D000199:Actins; D020409:Molecular Motor Proteins; D024462:Myosin Type II; D024701:Myosin Type V