The kinetic mechanism of mouse myosin VIIA

Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s(-1) is activated by actin only by ～3.5-fold resulting in a V(max) of 0.7 s(-1) and a K(ATPase) of 11.5 μM. There is no single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M·T M·D·P) is slow (12 s(-1)) and the equilibrium constant (K(H)) of 1 suggests significant reversal of hydrolysis. In the presence of actin ADP dissociates with a rate constant of 1.2 s(-1). Phosphate dissociation is relatively fast (>12 s(-1)), but the maximal rate could not be experimentally obtained at actin concentrations ≤ 50 μM because of the weak binding of the myosin VIIa-ADP-P(i) complex to actin. At higher actin concentrations the rate of attached hydrolysis (0.4 s(-1)) becomes significant and partially rate-limiting. Our findings suggest that the myosin VIIa is a "slow", monomeric molecular motor with a duty ratio of 0.6.     

Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism

Mutations of myosin VIIA cause deafness in various species from human and mice to Zebrafish and Drosophila. We analyzed the kinetic mechanism of the ATPase cycle of Drosophila myosin VIIA by using a single-headed construct with the entire neck domain. The steady-state ATPase activity (0.06 s(-1)) was markedly activated by actin to yield V(max) and K(ATPase) of 1.72 s(-1) and 3.2 microm, respectively. The most intriguing finding is that the ATP hydrolysis predominantly takes place in the actin-bound form (actin-attached hydrolysis) for the actomyosin VIIA ATPase reaction. The ATP hydrolysis rate was much faster for the actin-attached form than the dissociated form, in contrast to other myosins reported so far. Both the ATP hydrolysis step and the phosphate release step were significantly faster than the entire ATPase cycle rate, thus not rate-determining. The rate of ADP dissociation from actomyosin VIIA was 1.86 s(-1), which was comparable with the overall ATPase cycle rate, thus assigned to be a rate-determining step. The results suggest that Drosophila myosin VIIA spends the majority of the ATPase cycle in an actomyosin.ADP form, a strong actin binding state. The duty ratio calculated from our kinetic model was approximately 0.9. Therefore, myosin VIIA is classified to be a high duty ratio motor. The present results suggested that myosin VIIA can be a processive motor to serve cargo trafficking in cells once it forms a dimer structure.     

Characterization of the motor activity of mammalian myosin VIIA

Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg(2+)-ATPase activity that was markedly activated by actin with K(actin) of 33 microm and V(max) of 0.53 s(-1) head(-1). Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K(actin) of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor.     

Human myosin Vc is a low duty ratio, nonprocessive molecular motor

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.     

Kinetic characterization of a monomeric unconventional myosin V construct.

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.     

Human myosin III is a motor having an extremely high affinity for actin

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.     

Kinetic mechanism of the fastest motor protein, Chara myosin

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 actin.motor 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.     

Kinetic mechanism of human myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.     

Mechanism of action of myosin X, a membrane-associated molecular motor

We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin.ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.     

Mechanoenzymatic characterization of human myosin Vb

There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V(max) and K(actin) of the actin-activated ATPase activity of HuM5B were 9.7 +/- 0.4 s(-)(1) and 8.5 +/- 0.1 microM, respectively. K(actin) and K(ATP) of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 +/- 0.5 s(-)(1), which was comparable to the V(max) of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 +/- 0.03 microm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.     

Temperature dependence of nucleotide association and kinetic characterization of myo1b

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. It is one of the best characterized myosin-I isoforms and appears to have unique biochemical properties tuned for tension sensing or tension maintenance. We determined the key biochemical rate constants that define the actomyo1b ATPase cycle at 37 degrees C and measured the temperature dependence of ATP binding, ADP release, and the transition from a nucleotide-inaccessible state to a nucleotide-accessible state (k(alpha)). The rate of ATP binding is highly temperature sensitive, with an Arrhenius activation energy 2-3-fold greater than other characterized myosins (e.g., myosin-II and myosin-V). ATP hydrolysis is fast, and phosphate release is slow and rate limiting with an actin dependence that is nearly identical to the steady-state ATPase parameters (Vmax and K(ATPase)). ADP release is not as temperature dependent as ATP binding. The rates and temperature dependence of ADP release are similar to k(alpha) suggesting that a similar structural change is responsible for both transitions. We calculate a duty ratio of 0.08 based on the biochemical kinetics. However, this duty ratio is likely to be highly sensitive to strain.     

Human myosin Vc is a low duty ratio nonprocessive motor

There are three distinct members of the myosin V family in vertebrates, and each isoform is involved in different membrane trafficking pathways. Both myosin Va and Vb have demonstrated that they are high duty ratio motors that are consistent with the processive nature of these motors. Here we report that the ATPase cycle mechanism of the single-headed construct of myosin Vc is quite different from those of other vertebrate myosin V isoforms. K(ATPase) of the actin-activated ATPase was 62 microm, which is much higher than that of myosin Va ( approximately 1 mum). The rate of ADP release from actomyosin Vc was 12.7 s(-1), which was 2 times greater than the entire ATPase cycle rate, 6.5 s(-1). P(i) burst size was 0.31, indicating that the equilibrium of the ATP hydrolysis step is shifted to the prehydrolysis form. Our kinetic model, based on all kinetic data we determined in this study, suggests that myosin Vc spends the majority of the ATPase cycle time in the weak actin binding state in contrast to myosin Va and Vb. Consistently, the two-headed myosin Vc construct did not show processive movement in total internal reflection fluorescence microscope analysis, demonstrating that myosin Vc is a nonprocessive motor. Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms.     

The kinetic mechanism of Myo1e (human myosin-IC)

Myo1e is the widely expressed subclass-1 member of the myosin-I family. We performed a kinetic analysis of a truncated myo1e that consists of the motor and the single IQ motif with a bound calmodulin. We determined the rates and equilibrium constants for the key steps in the ATPase cycle. The maximum actin activated ATPase rate (V(max)) and the actin concentration at half-maximum of V(max) (K(ATPase)) of myo1e are similar to those of the native protein. The K(ATPase) is low (approximately 1 microm), however the affinity of myo1e for actin in the presence of ATP is very weak. A weak actin affinity and a rapid rate of phosphate release result in a pathway under in vitro assay conditions in which phosphate is released while myo1e is dissociated from actin. Actin activation of the ATPase activity and the low K(ATPase) are the result of actin activation of ADP release. We propose that myo1e is tuned to function in regions of high concentrations of cross-linked actin filaments. Additionally, we found that ADP release from actomyo1e is > 10-fold faster than other vertebrate myosin-I isoforms. We propose that subclass-1 myosin-Is are tuned for rapid sliding, whereas subclass-2 isoforms are tuned for tension maintenance or stress sensing.     

Kinetic mechanism and regulation of myosin VI.

Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of P(i) release, which lowers the K(ATPase) but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins.     

Glu257 in GroEL is a sensor involved in coupling polypeptide substrate binding to stimulation of ATP hydrolysis

The ATPase activity of many types of molecular chaperones is stimulated by polypeptide substrate binding via molecular mechanisms that are, for the most part, unknown. Here, we report that such stimulation of the ATPase activity of GroEL is abolished when its conserved apical domain residue Glu257 is replaced by alanine. This mutation is also found to convert the ATPase profile of GroEL, a group I chaperonin, into one that is characteristic of group II chaperonins. Steady-state and transient kinetic analysis indicate that both effects are due, at least in part, to a reduction of the affinity of GroEL for ADP. This finding indicates that nonfolded proteins stimulate ATP hydrolysis by accelerating the off-rate of the ADP formed, thereby allowing more rapid cycles of ATP binding and hydrolysis.     

Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIb

We examined the effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1). Blebbistatin is a small molecule that affects cell blebbing during the process of cell division, which has been shown to decrease the myosin ATPase activity of a number of myosins [Straight et al. (2003) Science 299, 1743-1747]. The steady-state actin-activated ATPase activity of NMIIB S1 was decreased approximately 90% at 40 microM actin in the presence of blebbistatin. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis. Binding to actin in the presence of ADP (0.57 +/-0.08 microM(-1) s(-1)) was reduced slightly in the presence of blebbistatin (0.38 +/- 0.03 microM(-1) s(-1)), while mantADP dissociation from acto-NMIIB S1 was reduced (approximately 30%). P(i) release was blocked in the presence of blebbistatin. Accordingly, the apparent affinity of NMIIB S1 for actin in the presence of ATP was greatly reduced. Based on the above data, we surmise that blebbistatin inhibits the ATPase activity of NMIIB S1 primarily by blocking entry into the strong binding state; secondarily, it reduces the rate of ADP release. These effects are likely mediated by binding of blebbistatin within the myosin cleft that progressively closes in forming the acto-myosin rigor state.     

Kinetics processivity and the direction of motion of Ncd.

The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209-700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s(-1) is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K(d) values are 1.0 microM for ADP-AlF(4), 1.1 microM for ATPgammaS, 1.5 microM for ATP, 3 microM for ADP, and 10 microM for ADP-vanadate (in 25 mM NaCl, 22 degrees C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 microM(-1) s(-1). Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme,[see text] where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k(1) and k(-4) are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1-mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1.ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.     

Myosin Va becomes a low duty ratio motor in the inhibited form

Vertebrate myosin Va is a typical processive motor with high duty ratio. Recent studies have revealed that the actin-activated ATPase activity of the full-length myosin Va (M5aFull) is inhibited at a low [Ca(2+)], which is due to the formation of a folded conformation of M5aFull. To clarify the underlying inhibitory mechanism, we analyzed the actin-activated ATP hydrolysis mechanism of the M5aFull at the inhibited and the activated states, respectively. Marked differences were found in the hydrolysis, P(i) release, and ADP release steps between the activated and the inhibited states. The kinetic constants of these steps of the activated state were similar to those of the unregulated S1 construct, in which the rate-limiting step was the ADP release step. On the other hand, the P(i) release rate from acto-M5aFull was decreased in EGTA by >1,000-fold, which makes this step the rate-limiting step for the actin-activated ATP hydrolysis cycle of M5aFull. The ADP off rate from acto-M5aFull was decreased by approximately 10-fold, and the equilibrium between the prehydrolysis state and the post hydrolysis state was shifted toward the former state in the inhibited state of M5aFull. Because of these changes, M5aFull spends a majority of the ATP hydrolysis cycling time in the weak actin binding state. The present results indicate that M5aFull molecules at a low [Ca(2+)] is inhibited as a cargo transporter not only due to the decrease in the cross-bridge cycling rate but also due to the decrease in the duty ratio thus being dissociated from actin.     

Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.     

Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.