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...

The light chain binding domain of rat myosin 1d consists of two IQ‐motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ‐motifs and Ca2+/CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d‐head, Myo1d‐IQ1, Myo1d‐IQ1.2, Myo1d‐IQ2 and Myo1dΔLV‐IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2+. IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+. The actin‐activated ATPase activity of Myo1d was ∼75% inhibited by Ca2+‐binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2+‐dependence, we propose that Ca2+‐binding to the C‐terminal pair of high affinity sites in CaM inhibits the Myo1d actin‐activated ATPase activity. This inhibition was due to a conformational change of the C‐terminal lobe of CaM remaining bound to the IQ‐motif(s). Interestingly, a similar but Ca2+‐independent inhibition of Myo1d actin‐activated ATPase activity was observed when IQ2, fused directly to the Myo1d‐head, was rotated through 200° by the deletion of two amino acids in the lever arm α‐helix N‐terminal to the IQ‐motif.