Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin.
Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected V(max) and K(app) of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected K(app) and the velocity, although it did not affect V(max). Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.
Ito
Kohji
K
Department of Biology, Chiba University, Inage-ku, Chiba City, Chiba 263-8522, Japan. k-ito@faculty.chiba-u.jp
Yamaguchi
Yukie
Y
Yanase
Kenji
K
Ichikawa
Yousuke
Y
Yamamoto
Keiichi
K
D000595:Amino Acid Sequence; D000818:Animals; D044784:Chara / Q000737:chemistry*; D002645:Chickens; D007700:Kinetics; D008969:Molecular Sequence Data; D009218:Myosins / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D013499:Surface Properties
D009218:Myosins