Functional Role of the Extended Loop 2 in the Myosin 9b Head for Binding F-actin

Abstract

The mammalian class IX myosin Myo9b can move considerable distances along actin filaments before it dissociates. This is remarkable, because it is single headed and because the ratelimiting step in its ATPase cycle is ATP hydrolysis. Thus, it spends most of its cycling time in the ATP-bound state that has a weak affinity for F-actin in other myosins. It has been speculated that the very extended loop 2 in the Myo9b head domain comprises an additional actin-binding site that prevents it from dissociation in the weak binding states. Here we show that two regions in the loop 2 determine the F-actin concentrations needed to maximally activate the steady-state ATPase activity. Together these two regions regulate the amount capable of binding F-actin and the affinity of the nucleotide-free state. The isolated loop 2 behaved like an entropic spring and bound stoichiometrically and with high affinity to F-actin. Subfragment 1 from skeletal muscle myosin II bound to F-actin simultaneously with the isolated loop 2 of Myo9b and could not displace it. Furthermore, the present results imply also a regulatory role for the tail region. Taken together, the results demonstrate that the extended loop 2 in Myo9b binds F-actin and influences the binding of the conventional stereo-specific actin-binding site.Myosin 9b (Myo9b, myr 5) 2 has been reported to move processively along actin filaments, i.e. upon binding to an actin filament it takes multiple consecutive steps before it dissociates (1-3). This is remarkable, because Myo9b is a single-headed myosin. Other myosins that move processively are two-headed and coordinate movement between the two heads (4, 5). Myo9b is also unique in that ATP hydrolysis is the rate-limiting step in the ATPase cycle (6, 7). This means that Myo9b spends a considerable amount of its cycling time in the ATP-bound state that represents a typically weak actin affinity state. However, Myo9b in the ATP-bound state binds with a relatively high affinity to F-actin (6, 7). Nevertheless, kinetic data do not unequivocally support processive movement.It has been speculated that the exceptionally long insertion at the position of loop 2 in the myosin head tethers Myo9b to F-actin and prevents it from diffusing away. The loop 2 in myosins is a surface loop that has been implicated in the initial weak electrostatic interaction with F-actin. In the processive myosin Va this loop is a little longer and more positively charged than in the non-processive class II myosins. An increase in the net positive charge of loop 2 increased the affinity of myosin Va for F-actin in all nucleotide states, whereas a decrease in its net positive charge reduced the affinity (8). Similar findings were also obtained with naturally occurring splice variants of the non-processive myosin V from Drosophila melanogaster and by modifying the loop 2 in class II myosins (8 -12). The processive run length of myosin Va varied with the affinity for F-actin in the weak binding states (13,14). A higher net positive charge of loop 2 increased the pro...

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

“…It could e.g. influence the interaction with F-actin, as the loop 2 in rat Myo9b has been shown to regulate the interaction with actin filaments (21). In class VI myosin, calmodulin was found to be associated with a unique insert between the converter and the IQ-motif that repositions the lever arm and reverses directional movement of myosin VI toward the minus-end of actin filaments (37,38).…”

“…Interestingly, addition of free Ca 2ϩ increased the basal ATPase activity of the Myo9-head-4IQ and accelerated actin filament gliding. A comparison of the steady-state ATPase activity and the velocity in the gliding assay of the C. elegans Myo9-head-4IQ protein with those of analogous or similar mammalian Myo9b constructs revealed a remarkable similarity (6,20,21). However, mammalian Myo9b constructs were analyzed in the absence of Ca 2ϩ only.…”

“…Indeed, Myo9b binds to F-actin with a relatively high affinity in the ATP-bound state that for other myosins represents a weak actin-binding state (20,22,23). The critical importance of the unique insertion for the binding of Myo9b to F-actin has been further demonstrated by the findings that deletion of the insertion lowers F-actin affinity considerably and that the isolated loop 2 insertion of Myo9b binds to F-actin with high affinity (21,22). In the two-headed processive motors the release of ADP is rate-limiting so that these myosins spend most of their cycling time in the high F-actin affinity ADP-bound state.…”

“…A similar strategy was applied to obtain the Myo9-head construct encompassing amino acids 1-959 of C. elegans Myo9 followed by three alanine residues, the 15 residues Avi tag, three glycine residues, and the FLAG tag. Recombinant baculovirus DNA was generated by the Bac-to-Bac method as described previously and transfected into Spodoptera frugiperda (Sf9) cells (21). Individual viruses were isolated by end point dilution and then amplified three times.…”

Functional Characterization of Human Myosin-18A and Its Interaction with F-actin and GOLPH3

“…described previously (22)(23)(24)(25). Averaged data of replicate binding experiments using three different preparations are given in the figures.…”