Kinetic Mechanism of the Fastest Motor Protein, Chara Myosin

Abstract

Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin⅐motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s ؊1 , and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s ؊1 . From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a V max value of the actin-activated ATPase activity of 390 s ؊1 . The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate.Myosin is an actin-based motor protein that converts chemical energy liberated by the ATP hydrolysis into directed movement of actin filaments. Phylogenetic analysis of myosin sequences revealed that there are at least 24 classes of myosin (1). Motor functions, such as motility and ATP hydrolysis activity, vary significantly among these classes of myosin. Among those known so far, myosin from alga Chara corallina is the fastest, moving actin filaments at 40 -60 m/s in the in vitro motility assay (2-4). This velocity is ϳ10 times faster than that of the fast skeletal muscle myosin (5). We have been interested in the mechanism how this plant myosin can move so fast. To understand the molecular mechanism of the fast movement,

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

“…As mentioned earlier, the rate-limiting step of acto-Chara myosin ATPase is suggested to be the transition from a weekly bound state with actin to a strongly bound state with concomitant release of Pi (6). Because substitution of positively charged amino acids in loop 3 to alanine lowered V max , the interaction with actin through loop 3 seems to play a critical role in triggering the transition from the weakly to the strongly bound state.…”

“…Actin-activated ATPase activity of expressed Chara motor domain was approximately 500 s Ϫ1 head Ϫ1 , which was also the highest of all myosins so far measured (5). Since the ADP release and ATP-induced dissociation was very fast as mentioned above, the rate-limiting step of acto-Chara myosin ATPase is suggested to be the transition from the weekly to the strongly bound state with actin with concomitant release of Pi (6).…”

“…Relevant steps in actomyosin ATPase cycle to the sliding velocity are ADP release from actomyosin and ATP reassociation resulting in actin-myosin dissociation. Kinetic analyses of Chara myosin motor domain revealed that both the ADP release and the ATP-induced dissociation from actin were very fast (6). Time spent in the strongly bound state with actin, which was estimated from these rates, was less than 1 ms.…”

“…Chara myosin motor domains used in this study contain only motor domain but not IQ motifs while native Chara myosin has 6 IQ motifs, lever arm length, and thus the velocity of the recombinant Chara myosin motor domains, are about one-eighth of those of the native Chara myosin (5,6). Loops 2 and 3 of the motor domain were mutated by PCR-based mutagenesis.…”

“…This velocity is about 10 times faster than that of the fast skeletal muscle myosin and the Chara myosin is the fastest motor protein known so far. We have cloned cDNA of the Chara myosin heavy chain (4) and succeeded in expressing functional motor domain (5,6). The velocity of the expressed motor domain measured by in vitro motility assay was comparable to that of the native Chara myosin if we consider the difference in the lever arm length.…”